Tumor selective macropinocytosis-dependent rapidly internalizing antibodies

ABSTRACT

Methods are provided for identifying and selecting antibodies that are internalized into cells via the macropinocytosis pathway. Additionally antibodies that are internalized via this pathway are provided as well as immunoconjugates comprising such antibodies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. 371 National phase of PCT/US2015/039741,filed Jul. 9, 2015, which claims benefit of and priority to USSN62/023,689, filed on Jul. 11, 2014, both of which are incorporatedherein by reference in their entirety for all purposes.

STATEMENT OF GOVERNMENTAL SUPPORT

This invention was made with government support under Grants No. R01CA118919, R01 CA129491 and R01 CA171315 awarded by the NationalInstitutes of Health. The Government has certain rights in thisinvention.

BACKGROUND

There is significant interest in the development of targetedtherapeutics such as antibody drug conjugates that have the potential toimprove the therapeutic window of cytotoxic drugs by delivering themspecifically and intracellularly to cancer cells (Austin et al. (2004)Mol. Biol. Cell. 15: 5268-5282; Burris et al. (2011) Clin. BreastCancer. 11: 275-282; Sievers and Senter (2013) Annu. Rev. Med. 64:15-29; Behrens and Liu (2013) MAbs, 6(1): 46-53; Sutherland et al.(2006) J. Biol. Chem. 281: 10540-10547). The pathway by which thetargeted agent enters tumor cells can influence both the uptakeefficiency and the intracellular fate of the internalized agent, both ofwhich contribute to the cytotoxic potency (Sutherland et al. (2006) J.Biol. Chem. 281: 10540-10547; Erickson et al. (2006) Cancer Res. 66:4426-4433).

Endocytosis pathways can be subdivided into four categories: 1)clathrin-mediated endocytosis, 2) caveolae, 3) macropinocytosis, and 4)phagocytosis. Clathrin-mediated endocytosis is mediated by small(approx. 100 nm in diameter) vesicles that have a morphologicallycharacteristic coat made up of a complex of proteins that are mainlyassociated with the cytosolic protein clathrin. Clathrin-coated vesicles(CCVs) are found in virtually all cells and form domains of the plasmamembrane termed clathrin-coated pits. Coated pits can concentrate largeextracellular molecules that have different receptors responsible forthe receptor-mediated endocytosis of ligands, e.g. low densitylipoprotein, transferrin, growth factors, antibodies and many others.

Caveolae are the most common reported non-clathrin-coated plasmamembrane buds, which exist on the surface of many, but not all celltypes. They consist of the cholesterol-binding protein caveolin (Vip21)with a bilayer enriched in cholesterol and glycolipids. Caveolae aresmall (approximately 50 nm in diameter) flask-shape pits in the membranethat resemble the shape of a cave (hence the name caveolae). They canconstitute up to a third of the plasma membrane area of the cells ofsome tissues, being especially abundant in smooth muscle, type Ipneumocytes, fibroblasts, adipocytes, and endothelial cells (Burris etal. (2011) Clin. Breast Cancer. 11: 275-282). Uptake of extracellularmolecules is also believed to be specifically mediated via receptors incaveolae.

Macropinocytosis, which usually occurs from highly ruffled regions ofthe plasma membrane, is the invagination of the cell membrane to form apocket, which then pinches off into the cell to form a vesicle (˜0.5-5μm in diameter) filled with a large volume of extracellular fluid andmolecules within it (equivalent to ˜100 CCVs). The filling of the pocketoccurs in a non-specific manner. The vesicle then travels into thecytosol and fuses with other vesicles such as endosomes and lysosomes.

Phagocytosis is the process by which cells bind and internalizeparticulate matter larger than around 0.75 μm in diameter, such assmall-sized dust particles, cell debris, micro-organisms and evenapoptotic cells, which only occurs in specialized cells. These processesinvolve the uptake of larger membrane areas than clathrin-mediatedendocytosis and caveolae pathway.

SUMMARY

Macropinocytosis was investigated as an intriguing pathway for cellularentry because it is a form of bulk uptake and can therefore efficientlyand rapidly internalize targeting agents. Macropinosomes comprise large,endocytic vesicles that range from 0.2 μm to 3 μm in size, which are upto 30-fold larger than the 0.1 μm average size of protein-coated,endocytic vesicles (Hewlett et al. (1994) J. Cell Biol. 124: 689-703).Additionally, studies have shown that macropinocytosis is selectivelyupregulated in Ras-transformed cancers (a common oncogenic mutation inhuman cancers) and plays an important role in tumor cell homeostasis byserving as an amino acid supply route (Commisso et al. (2013) Nature.497: 633-637), suggesting that targeted therapeutics based on antibodiesthat internalize via the macropinocytosis pathway may provide additionaltumor-specificity against a wide variety of human cancers.

To therapeutically explore the utility of antibodies that gain entryinto tumor cells via receptor-dependent macropinocytosis, a generallyapplicable method was developed that readily identifies such antibodies.While phage antibody display libraries have been extensively used toselect for antibodies that internalize into tumor cells, it is believedthat no methods have been previously developed to uncover antibodiescapable of cellular entry through the macropinocytosis pathway.

To this end, a high content analysis (HCA)-based screening strategy wasdeveloped that employs automated image-based analysis to identify phageantibodies that colocalize with a macropinocytosis marker (e.g., TexasRed-conjugated 70 kDa neutral dextran (ND70-TR)). The HCA protocol wasused to screen single chain variable fragment (scFv) phage antibodydisplay libraries that were previously generated by laser capturemicrodissection (LCM)-based selection on live tumor cells and tumortissues, which are highly enriched for internalizing phage antibodiesbinding to prostate tumor cells in situ residing in their tissuemicroenvironment (Ruan et al. (2006) Mol. Cell Proteomics. 5:2364-2373), and identified antibodies that are capable of efficientinternalization via macropinocytosis. Kinetics and subcellularcolocalization studies were performed for phage antibodies as well asfull-length immunoglobulin G (IgG) molecules derived from the parentalscFvs and identified a highly active, macropinocytosing antibody thatrapidly internalizes and colocalizes with early endosomal and lysosomalmarkers. The target antigen was identified as EphA2 byimmunoprecipitation and mass spectrometry. To confirm internalization byan independent functional assay and to demonstrate therapeuticpotential, an antibody-toxin conjugate was created and it showed potentand specific cytotoxic activity against a panel of EphA2-positive tumorcell lines. It is believed this is the first description of a generallyapplicable screening strategy to uncover macropinocytosing antibodies,enabling further exploration of this class of antibody-antigen pairs forthe development of effective antibody-targeted therapeutics.

Various embodiments contemplated herein may include, but need not belimited to, one or more of the following:

Embodiment 1: A method of preparing antibodies that are internalizedinto a cell by a macropinocytosis pathway, said method comprising:

-   -   contacting target cells with members of an antibody library and        with a marker for macropinocytosis;    -   identifying internalized antibodies that co-localize in said        target cells with said marker for macropinocytosis; and    -   selecting those antibodies that co-localize with said marker for        macropinocytosis.

Embodiment 2: The method of embodiment 1, wherein said members of anantibody library are members of a phage display library.

Embodiment 3: The method of embodiment 1, wherein said members of anantibody library are members of a yeast display library.

Embodiment 4: The method according to any one of embodiments 1-3,wherein said antibody library is an antibody library that is enrichedfor antibodies that bind to tumor cells.

Embodiment 5: The method of embodiment 4, wherein said antibody libraryis an antibody library that is enriched for antibodies that bind totumor cells and said enrichment is by laser capture microdissection(LCM) of antibodies that bind to tumor cells.

Embodiment 6: The method according to any one of embodiments 1-5,wherein said antibody library is an antibody library that is enrichedfor antibodies that are internalized into tumor cells.

Embodiment 7: The method according to any one of embodiments 1-6,wherein said marker for macropinocytosis includes a marker selected fromthe group consisting of high molecular weight dextran, latex beads,glass beads, Lucifer yellow, and soluble enzymes such as horseradishperoxidase.

Embodiment 8: The method of embodiment 7, wherein said marker formacropinocytosis includes labeled high molecular weight dextran.

Embodiment 9: The method of embodiment 8, wherein said marker formacropinocytosis includes labeled high molecular weight dextran having amolecular weight that ranges from about 60 kDa to about 80 kDa.

Embodiment 10: The method of embodiment 8, wherein said marker formacropinocytosis includes labeled high molecular weight dextran having amolecular weight of about 70 kDa.

Embodiment 11: The method of embodiment 7, wherein, wherein said markerfor macropinocytosis includes latex beads or glass beads.

Embodiment 12: The method of embodiment 11, wherein said latex beads orglass beads are approximately 20 nm in diameter.

Embodiment 13: The method according to any one of embodiments 1-12,wherein said marker for macropinocytosis is labeled with a detectablelabel.

Embodiment 14: The method of embodiment 13, wherein said marker formacropinocytosis is labeled with a fluorescent label.

Embodiment 15: The method of embodiment 14, wherein said marker formacropinocytosis is labeled with fluorescein isothiocyanate (FITC) ortetrarhodamine isothiocyanate (TRITC).

Embodiment 16: The method of embodiment 7, wherein, wherein said markerfor macropinocytosis includes Lucifer yellow.

Embodiment 17: The method according to any one of embodiments 1-16,wherein said target cells comprise cells of tumor cell lines.

Embodiment 18: The method of embodiment 17, wherein said target cellsare selected from the group consisting of PC3, DU145, HeLa, MDA-MB-231,Hs5786, MDA-435, BT549, SKOV3, HeyA8, OVCAR3, PANC1, MIAPaCa2, BxPC3,T24, TCCSUP, UMUC-3, TE1, AGS, SGC-7901, M28, VAMT-1, A549, A431,A172MG, DBTRG-5MG, U-251MG, U87MG, T84, THP1, U373, U937, VCaP, SiHa,FM3, DuCaP, A253, A172, 721, SiHa, and LNCaP.

Embodiment 19: The method according to any one of embodiments 1-18,wherein said contacting includes incubating said members of an antibodylibrary and/or said marker for macropinocytosis with said cells.

Embodiment 20: The method according to any one of embodiments 1-19,wherein said contacting includes incubating said members of an antibodylibrary and/or said marker for macropinocytosis with said cells for aperiod of at least 1 hour, or at least 2 hours, or at least 3 hours, orat least 4 hours, or at least 6 hours, or at least 8 hours, or at least10 hours, or at least 12 hours, or at least 16 hours, or at least 20hours, or at least 24 hours.

Embodiment 21: The method according to any one of embodiments 1-19,wherein said identifying includes high content screening (HCS) of saidcells.

Embodiment 22: The method of embodiment 21, wherein said high contentscreening is performed using a fluorescent microscope and automateddigital microscopy.

Embodiment 23: The method according to any one of embodiments 1-22,wherein said colocalized antibody is labeled with a fluorescent labelattached to a second antibody that binds said colocalized antibody.

Embodiment 24: The method of embodiment 23, wherein said second antibodyincludes an anti-fd bacteriophage.

Embodiment 25: The method according to any one of embodiments 1-24,wherein said method further includes selecting internalized antibodiesthat colocalize with a lysosomal marker.

Embodiment 26: The method of embodiment 25, wherein said antibodycolocalizes with LAMP1.

Embodiment 27: The method according to any one of embodiments 1-26,wherein said selecting comprises recovering the antibody from the sampleused in the HCS analysis.

Embodiment 28: The method according to any one of embodiments 1-26,wherein said selecting comprises selecting the antibodies from thelibrary corresponding to the antibodies identified in the HCS analysis.

Embodiment 29: The method according to any one of embodiments 1-28,wherein said selecting comprises determining the amino acid sequence ofsaid antibody.

Embodiment 30: The method according to any one of embodiments 1-29,wherein said selecting comprises converting said antibody into an intactimmunoglobulin.

Embodiment 31: The method of embodiment 30, wherein said selectingcomprises converting said antibody into an IgG.

Embodiment 32: The method of embodiment 30, wherein said selectingcomprises converting said antibody into an IgA.

Embodiment 33: An isolated antibody that is internalized into a cell viaa macropinocytosis pathway, wherein said antibody is an antibody thatbinds to ephrin type A receptor 2 (EphA2).

Embodiment 34: The antibody of embodiment 33, wherein said antibody isan antibody that is identified using the method of embodiments 1-32.

Embodiment 35: The antibody according to any one of embodiments 33-34,wherein said antibody is a human antibody.

Embodiment 36: The antibody according to any one of embodiments 33-35,wherein said antibody is an antibody selected from the group consistingof an intact immunoglobulin, a Fab, a (Fab′)₂, an scFv, and an (ScFv′)₂.

Embodiment 37: The antibody of embodiment 36, wherein said antibody isan intact immunoglobulin.

Embodiment 38: The antibody of embodiment 37, wherein said antibody isan IgG or an IgA.

Embodiment 39: The antibody according to any one of embodiments 33-38,wherein said antibody is a monoclonal antibody.

Embodiment 40: The antibody according to any one of embodiments 33-39,wherein said antibody is internalized via a macropinocytosis pathway ina cell in which macropinocytosis is upregulated.

Embodiment 41: The antibody of embodiment 40, wherein said cell is acancer cell.

Embodiment 42: The antibody of embodiment 41, wherein said cell is aRas-transformed cancer cell.

Embodiment 43: The antibody of embodiment 41, wherein said cell is acancer cell selected from the group consisting of PC3, DU145, HeLa,MDA-MB-231, Hs5786, MDA-435, BT549, SKOV3, HeyA8, OVCAR3, PANC1,MIAPaCa2, BxPC3, T24, TCCSUP, UMUC-3, TE1, AGS, SGC-7901, M28, VAMT-1,A549, A431, A172MG, DBTRG-5MG, U-251MG, U87MG, T84, THP1, U373, U937,VCaP, SiHa, FM3, DuCaP, A253, A172, 721, SiHa, and LNCaP.

Embodiment 44: The antibody according to any one of embodiments 33-43,wherein said antibody competes with one or more antibodies selected fromthe group consisting of HCA-F1, and HCA-F2 for binding EphA2.

Embodiment 45: The antibody of embodiment 44, wherein said antibodycompetes with HCA-F1 for binding EphA2.

Embodiment 46: The antibody of embodiment 44, wherein said antibodycompetes with HCA-F2 for binding EphA2.

Embodiment 47: The antibody of embodiment 44, wherein said antibodybinds the same epitope bound by HCA-F1.

Embodiment 48: The antibody of embodiment 44, wherein said antibodybinds the same epitope bound by HCA-F2.

Embodiment 49: The antibody according to any one of embodiments 33-48,wherein said antibody includes VH CDR1, VH CDR2, and VH CDR3 of HCA-F1.

Embodiment 50: The antibody according to any one of embodiments 33-48,or embodiment 49, wherein said antibody includes VL CDR1, VL CDR2, andVL CDR3 of HCA-F1.

Embodiment 51: The antibody according to any one of embodiments 33-48,wherein said antibody includes VH CDR1, VH CDR2, and VH CDR3 of HCA-F2.

Embodiment 52: The antibody according to any one of embodiments 33-48,or embodiment 51, wherein said antibody includes VL CDR1, VL CDR2, andVL CDR3 of HCA-F2.

Embodiment 53: The antibody according to any one of embodiments 33-48,wherein said antibody includes the VH and/or the VL domain of HCA-F1.

Embodiment 54: The antibody of embodiment 53, wherein said antibodyincludes the VH and the VL domain of HCA-F1.

Embodiment 55: The antibody according to any one of embodiments 33-48,wherein said antibody includes the VH and/or the VL domain of HCA-F2.

Embodiment 56: The antibody of embodiment 55, wherein said antibodyincludes the VH and the VL domain of HCA-F2.

Embodiment 57: An isolated antibody that binds to a tumor cell.

Embodiment 58: The antibody of embodiment 57, wherein said antibody is ahuman antibody.

Embodiment 59: The antibody according to any one of embodiments 57-58,wherein said antibody is a monoclonal antibody.

Embodiment 60: The antibody according to any one of embodiments 57-59,wherein said cell is a cancer cell.

Embodiment 61: The antibody of embodiment 60, wherein said cell is acancer cell selected from the group consisting of PC3, DU145, HeLa,MDA-MB-231, Hs5786, MDA-435, BT549, SKOV3, HeyA8, OVCAR3, PANC1,MIAPaCa2, BxPC3, T24, TCCSUP, UMUC-3, TE1, AGS, SGC-7901, M28, VAMT-1,A549, A431, A172MG, DBTRG-5MG, U-251MG, U87MG, T84, THP1, U373, U937,VCaP, SiHa, FM3, DuCaP, A253, A172, 721, SiHa, and LNCaP.

Embodiment 62: The antibody according to any one of embodiments 57-61,wherein said antibody includes VH CDR1, VH CDR2, and VH CDR3 of HCA-M1.

Embodiment 63: The antibody according to any one of embodiments 57-61,or embodiment 62, wherein said antibody includes VL CDR1, VL CDR2, andVL CDR3 of HCA-M1.

Embodiment 64: The antibody according to any one of embodiments 57-61,wherein said antibody includes VH CDR1, VH CDR2, and VH CDR3 of HCA-S1.

Embodiment 65: The antibody according to any one of embodiments 57-61,or embodiment 64, wherein said antibody includes VL CDR1, VL CDR2, andVL CDR3 of HCA-S1.

Embodiment 66: The antibody according to any one of embodiments 57-61,wherein said antibody includes the VH and/or the VL domain of HCA-M1.

Embodiment 67: The antibody of embodiment 66, wherein said antibodyincludes the VH and the VL domain of HCA-M1.

Embodiment 68: The antibody according to any one of embodiments 57-61,wherein said antibody includes the VH and/or the VL domain of HCA-S1.

Embodiment 69: The antibody of embodiment 68, wherein said antibodyincludes the VH and the VL domain of HCA-S1.

Embodiment 70: The antibody according to any one of embodiments 33-69,wherein said antibody is a substantially intact immunoglobulin.

Embodiment 71: The antibody of embodiment 70, wherein said antibodyincludes an IgA, IgE, or IgG.

Embodiment 72: The antibody of embodiment 70, wherein said antibodyincludes an IgG1.

Embodiment 73: The antibody according to any one of embodiments 33-56,wherein said antibody is an antibody fragment selected from the groupconsisting of Fv, Fab, (Fab)₂, (Fab′)₃, IgGΔCH2, and a minibody.

Embodiment 74: The antibody according to any one of embodiments 33-69,wherein said antibody is a single chain antibody.

Embodiment 75: The antibody of embodiment 74, wherein the VL region ofsaid antibody is attached to the VH region of said antibody by an aminoacid linker ranging in length from about 3 amino acids up to about 15amino acids.

Embodiment 76: The antibody of embodiment 74, wherein the VL region ofsaid antibody is attached to the VH region of said antibody by linkerhaving the amino acid sequence (Gly₄Ser)₃SEQ ID NO:10.

Embodiment 77: An immunoconjugate including an antibody according to anyone of embodiments 33-76 attached to an effector wherein said effectoris selected from the group consisting of a second antibody, a detectablelabel, a cytotoxin or cytostatic agent, a liposome containing a drug, aradionuclide, a drug, a prodrug, a viral particle, a cytokine, achelate, and an siRNA.

Embodiment 78: The immunoconjugate of embodiment 77, wherein saidantibody is attached to an siRNA.

Embodiment 79: The immunoconjugate of embodiment 78, wherein said siRNAcarried by a liposome or mesoporous silica.

Embodiment 80: The immunoconjugate of embodiments 78-79, wherein saidsiRNA is an EphA2-targeted siRNA.

Embodiment 81: The immunoconjugate of embodiment 77, wherein saidantibody is attached to a cytotoxin.

Embodiment 82: The immunoconjugate of embodiment 81, wherein saidantibody is attached to a cytotoxin selected from the group consistingof a Diphtheria toxin, a Pseudomonas exotoxin, a ricin, an abrin,saporin, and a thymidine kinase.

Embodiment 83: The immunoconjugate of embodiment 77, wherein saidantibody is attached to a cytotoxic and/or cytostatic drug.

Embodiment 84: The immunoconjugate of embodiment 83, wherein saidantibody is attached directly or through a linker to one or more of thefollowing: said drug a lipid or liposome containing said drug; apolymeric drug carrier including said drug; and a nanoparticle drugcarrier including said drug.

Embodiment 85: The immunoconjugate according to any one of embodiments83-84, wherein said drug is an anti-cancer drug.

Embodiment 86: The immunoconjugate according to any one of embodiments83-84, wherein said drug is selected from the group consisting of atubulin inhibitor (e.g., auristatin, dolastatin, maytansine, colchicine,combretastatin, and the like), a DNA interacting agent (e.g.,calicheamicins, duocarmycins, pyrrolobenzodiazepines (PBDs), and thelike), and a pathway or enzyme inhibitor (e.g., mTOR/PI3K inhibitors,kinase and phosphatase inhibitors, RNA splicing inhibitors, RNApolymerase inhibitors, DNA polymerase inhibitors, topoisomeraseinhibitors, ribosome inhibitors, proteosome inhibitors, and the like).

Embodiment 87: The immunoconjugate according to any one of embodiments83-84, wherein said drug is selected from the group consisting ofauristatin, dolastatin, colchicine, combretastatin, and mTOR/PI3Kinhibitors.

Embodiment 88: The immunoconjugate according to any one of embodiments83-84, wherein said drug an auristatin is selected from the groupconsisting of Auristatin E (AE), Monomethylauristatin E (MMAE),Auristatin F (MMAF), vcMMAE, and vcMMAF.

Embodiment 89: The immunoconjugate according to any one of embodiments83-84, wherein said drug is monomethyl auristatin F.

Embodiment 90: The immunoconjugate of embodiment 89, wherein saidauristatin F is conjugated to said antibody via amaleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vcPAB)linker.

Embodiment 91: The immunoconjugate according to any one of embodiments83-84, wherein said drug is selected from the group consisting offlourouracil (5-FU), capecitabine, 5-trifluoromethyl-2′-deoxyuridine,methotrexate sodium, raltitrexed, pemetrexed, cytosine Arabinoside,6-mercaptopurine, azathioprine, 6-thioguanine (6-TG), pentostatin,fludarabine phosphate, cladribine, floxuridine (5-fluoro-2),ribonucleotide reductase inhibitor (RNR), cyclophosphamide, neosar,ifosfamide, thiotepa, 1,3-bis(2-chloroethyl)-1-nitosourea (BCNU),1,-(2-chloroethyl)-3-cyclohexyl-lnitrosourea, methyl (CCNU),hexamethylmelamine, busulfan, procarbazine HCL, dacarbazine (DTIC),chlorambucil, melphalan, cisplatin, carboplatin, oxaliplatin,bendamustine, carmustine, chloromethine, dacarbazine (DTIC),fotemustine, lomustine, mannosulfan, nedaplatin, nimustine,prednimustine, ranimustine, satraplatin, semustine, streptozocin,temozolomide, treosulfan, triaziquone, triethylene melamine, thioTEPA,triplatin tetranitrate, trofosfamide, uramustine, doxorubicin,daunorubicin citrate, mitoxantrone, actinomycin D, etoposide, topotecanHCL, teniposide (VM-26), irinotecan HCL (CPT-11), camptothecin,belotecan, rubitecan, vincristine, vinblastine sulfate, vinorelbinetartrate, vindesine sulphate, paclitaxel, docetaxel, nanoparticlepaclitaxel, abraxane, ixabepilone, larotaxel, ortataxel, tesetaxel, andvinflunine.

Embodiment 92: The immunoconjugate according to any one of embodiments83-84, wherein said drug is selected from the group consisting ofcarboplatin, cisplatin, cyclophosphamide, docetaxel, doxorubicin,erlotinib, etoposide, gemcitabine, imatinib mesylate, irinotecan,methotrexate, sorafinib, sunitinib, topotecan, vinblastine, andvincristine.

Embodiment 93: The immunoconjugate according to any one of embodiments83-84, wherein said drug is selected from the group consisting ofretinoic acid, a retinoic acid derivative, doxirubicin, vinblastine,vincristine, cyclophosphamide, ifosfamide, cisplatin, 5-fluorouracil, acamptothecin derivative, interferon, tamoxifen, and taxol. In certainembodiments the anti-cancer compound is selected from the groupconsisting of abraxane, doxorubicin, pamidronate disodium, anastrozole,exemestane, cyclophosphamide, epirubicin, toremifene, letrozole,trastuzumab, megestroltamoxifen, paclitaxel, docetaxel, capecitabine,goserelin acetate, and zoledronic acid.

Embodiment 94: The immunoconjugate of embodiment 77, wherein saidantibody is attached to a chelate including an isotope selected from thegroup consisting of ⁹⁹Tc, ²⁰³Pb, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ¹¹¹In, ¹¹³In, ⁹⁷Ru,⁶²Cu, ⁶⁴¹Cu, ⁵²Fe, ⁵²Mn, ⁵¹Cr, ¹⁸⁶Re, ¹⁸⁸Re, ⁷⁷As, ⁹⁰Y, ⁶⁷Cu, ¹⁶⁹Er,¹²¹Sn, ¹²⁷Te, ¹⁴²Pr, ¹⁴³Pr, ¹⁹⁸Au, ¹⁹⁹Au, ¹⁶¹Tb, ¹⁰⁹Pd, ¹⁶⁵Dy, ¹⁴⁹Pm,¹⁵¹Pm, ¹⁵³Sm, ¹⁵⁷Gd, ¹⁵⁹Gd, ¹⁶⁶Ho, ¹⁷²Tm, ¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁷⁷Lu, ¹⁰⁵Rh,and ¹¹¹Ag.

Embodiment 95: The immunoconjugate of embodiment 77, wherein saidantibody is attached to an alpha emitter.

Embodiment 96: The immunoconjugate of embodiment 95, wherein said alphaemitter is bismuth 213.

Embodiment 97: The immunoconjugate of embodiment 77, wherein saidantibody is attached to a lipid or a liposome complexed with orcontaining an anti-cancer drug.

Embodiment 98: The immunoconjugate of embodiment 77, wherein saidantibody is attached to a detectable label.

Embodiment 99: The immunoconjugate of embodiment 98, wherein saidantibody is attached to a detectable label selected from the groupconsisting of a radioactive label, a radio-opaque label, an MRI label,and a PET label.

Embodiment 100: A pharmaceutical formulation said formulation including:a pharmaceutically acceptable excipient and an antibody according to anyone of embodiments 33-76; and/or a pharmaceutically acceptable excipientand a immunoconjugate according to any one of embodiments 77-99.

Embodiment 101: The pharmaceutical formulation of embodiment 100,wherein said formulation is a unit dosage formulation.

Embodiment 102: The formulation according to any one of embodiments100-101, wherein said formulation is formulated for administration via aroute selected from the group consisting of oral administration, nasaladministration, rectal administration, intraperitoneal injection,intravascular injection, subcutaneous injection, transcutaneousadministration, and intramuscular injection.

Embodiment 103: A method of inhibiting the growth and/or proliferationof a cancer cell, said method including: contacting said cancer cellwith an antibody according to any one of embodiments 33-76 and/or animmunoconjugate according to any one of embodiments 77-99.

Embodiment 104: The method of embodiment 103, wherein said cancer cellis a cancer cell in which macropinocytosis is upregulated.

Embodiment 105: The method according to any one of embodiments 103-104,wherein said cancer cell is a Ras-transformed cancer cell.

Embodiment 106: The method according to any one of embodiments 103-105,wherein said cancer cell is selected from the group consisting ofovarian cancer, breast cancer, lung cancer, prostate cancer, coloncancer, kidney cancer, pancreatic cancer, mesothelioma, lymphoma, livercancer, urothelial cancer, melanoma, stomach cancer, and cervicalcancer.

Embodiment 107: The method according to any one of embodiments 103-106,wherein said cell is a metastatic cell.

Embodiment 108: The method according to any one of embodiments 103-107,wherein said cell is a solid tumor cell.

Embodiment 109: The method according to any one of embodiments 103-108,wherein said antibody and/or immunoconjugate is administered in apharmaceutical composition including a pharmaceutical acceptablecarrier.

Embodiment 110: The method according to any one of embodiments 103-109,wherein said administering includes administering to a human.

Embodiment 111: The method according to any one of embodiments 103-109,wherein said administering includes administering to a non-human mammal.

Embodiment 112: The method according to any one of embodiments 103-111,wherein said administering includes administering parenterally.

Embodiment 113: The method according to any one of embodiments 103-111,wherein said administering includes administering into a tumor or asurgical site.

Embodiment 114: The method according to any one of embodiments 103-113,wherein said immunoconjugate is administered as an adjunct therapy tosurgery and/or radiotherapy.

Embodiment 115: The method according to any one of embodiments 103-113,wherein said immunoconjugate is administered in conjunction with anotheranti-cancer drug and/or a hormone.

Embodiment 116: A method of detecting a cancer cell, said methodincluding: contacting said cancer cell with a immunoconjugate ofembodiment 99; and detecting the presence and/or location of saiddetectable label where the presence and/or location is an indicator ofthe location and/or presence of a prostate cancer cell.

Embodiment 117: The method of embodiment 116, wherein detecting includesa modality selected from the group consisting of said label includes alabel selected from the group consisting of a x-ray, CAT scan, MRI, PETscan, and radioimaging.

Embodiment 118: The method of embodiment 116, wherein said detectablelabel is selected from the group consisting of a gamma-emitter, apositron-emitter, an x-ray emitter, an alpha emitter, and afluorescence-emitter.

Embodiment 119: The method according to any one of embodiments 116-118,wherein said cancer cell is selected from the group consisting ofovarian cancer, breast cancer, lung cancer, prostate cancer, coloncancer, kidney cancer, pancreatic cancer, mesothelioma, lymphoma, livercancer, urothelial cancer, stomach cancer, and cervical cancer.

Embodiment 120: The method according to any one of embodiments 116-119,wherein said contacting includes administering said immunoconjugate to anon-human mammal.

Embodiment 121: The method according to any one of embodiments 116-119,wherein said contacting includes administering said immunoconjugate to ahuman.

Embodiment 122: The method according to any one of embodiments 120-121,wherein said detecting includes detecting said label in vivo.

Embodiment 123: A nucleic acid encoding an antibody or a fragment (e.g.,a binding fragment) of an antibody according to any of embodiments33-76.

Embodiment 124: An expression vector comprising the nucleic acid ofembodiment 123.

Embodiment 125: A cell containing the expression vector of embodiment124.

DEFINITIONS

As used herein, an “antibody” refers to a protein consisting of one ormore polypeptides substantially encoded by immunoglobulin genes orfragments of immunoglobulin genes. The recognized immunoglobulin genesinclude the kappa, lambda, alpha, gamma, delta, epsilon and mu constantregion genes, as well as myriad immunoglobulin variable region genes.Light chains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively.

A typical immunoglobulin (antibody) structural unit is known to comprisea tetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies exist as intact immunoglobulins or as a number of wellcharacterized fragments produced by digestion with various peptidases.Thus, for example, pepsin digests an antibody below the disulfidelinkages in the hinge region to produce F(ab)′₂, a dimer of Fab whichitself is a light chain joined to V_(H)—C_(H)1 by a disulfide bond. TheF(ab)′₂ may be reduced under mild conditions to break the disulfidelinkage in the hinge region thereby converting the (Fab′)₂ dimer into aFab′ monomer. The Fab′ monomer is essentially a Fab with part of thehinge region (see, Fundamental Immunology, W. E. Paul, ed., Raven Press,N.Y. (1993), for a more detailed description of other antibodyfragments). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchFab′ fragments may be synthesized de novo either chemically or byutilizing recombinant DNA methodology. Thus, the term antibody, as usedherein also includes antibody fragments either produced by themodification of whole antibodies or synthesized de novo usingrecombinant DNA methodologies. Preferred antibodies include single chainantibodies (antibodies that exist as a single polypeptide chain), morepreferably single chain Fv antibodies (sFv or scFv) in which a variableheavy and a variable light chain are joined together (directly orthrough a peptide linker) to form a continuous polypeptide. The singlechain Fv antibody is a covalently linked V_(H)-V_(L) heterodimer whichmay be expressed from a nucleic acid including V_(H)- and V_(L)-encodingsequences either joined directly or joined by a peptide-encoding linker.Huston, et al. (1988) Proc. Nat. Acad. Sci. USA, 85: 5879-5883. Whilethe V_(H) and V_(L) are connected to each as a single polypeptide chain,the V_(H) and V_(L) domains associate non-covalently. The firstfunctional antibody molecules to be expressed on the surface offilamentous phage were single-chain Fv's (scFv), however, alternativeexpression strategies have also been successful. For example Fabmolecules can be displayed on phage if one of the chains (heavy orlight) is fused to g3 capsid protein and the complementary chainexported to the periplasm as a soluble molecule. The two chains can beencoded on the same or on different replicons; the important point isthat the two antibody chains in each Fab molecule assemblepost-translationally and the dimer is incorporated into the phageparticle via linkage of one of the chains to, e.g., g3p (see, e.g., U.S.Pat. No. 5,733,743). The scFv antibodies and a number of otherstructures converting the naturally aggregated, but chemically separatedlight and heavy polypeptide chains from an antibody V region into amolecule that folds into a three dimensional structure substantiallysimilar to the structure of an antigen-binding site are known to thoseof skill in the art (see e.g., U.S. Pat. Nos. 5,091,513, 5,132,405, and4,956,778). Particularly preferred antibodies should include all thathave been displayed on phage (e.g., scFv, Fv, Fab and disulfide linkedFv (Reiter et al. (1995) Protein Eng. 8: 1323-1331).

The term “specifically binds”, as used herein, when referring to abiomolecule (e.g., protein, nucleic acid, antibody, etc.), refers to abinding reaction that is determinative of the presence biomolecule inheterogeneous population of molecules (e.g., proteins and otherbiologics). Thus, under designated conditions (e.g. immunoassayconditions in the case of an antibody or stringent hybridizationconditions in the case of a nucleic acid), the specified ligand orantibody binds to its particular “target” molecule and does not bind ina significant amount to other molecules present in the sample.

An “effector” refers to any molecule or combination of molecules whoseactivity it is desired to deliver/into and/or localize at cell.Effectors include, but are not limited to labels, cytotoxins, enzymes,growth factors, transcription factors, drugs, etc.

A “reporter” is an effector that provides a detectable signal (e.g. is adetectable label). In certain embodiments, the reporter need not providethe detectable signal itself, but can simply provide a moiety thatsubsequently can bind to a detectable label.

The term “conservative substitution” is used in reference to proteins orpeptides to reflect amino acid substitutions that do not substantiallyalter the activity (specificity or binding affinity) of the molecule.Typically, conservative amino acid substitutions involve substitution ofone amino acid for another amino acid with similar chemical properties(e.g. charge or hydrophobicity). The following six groups each containamino acids that are typical conservative substitutions for oneanother: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid(D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine(R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine(V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

The terms “epitope tag” or “affinity tag” are used interchangeablyherein, and used refers to a molecule or domain of a molecule that isspecifically recognized by an antibody or other binding partner. Theterm also refers to the binding partner complex as well. Thus, forexample, biotin or a biotin/avidin complex are both regarded as anaffinity tag. In addition to epitopes recognized in epitope/antibodyinteractions, affinity tags also comprise “epitopes” recognized by otherbinding molecules (e.g. ligands bound by receptors), ligands bound byother ligands to form heterodimers or homodimers, His₆ bound by Ni-NTA,biotin bound by avidin, streptavidin, or anti-biotin antibodies, and thelike.

Epitope tags are well known to those of skill in the art. Moreover,antibodies specific to a wide variety of epitope tags are commerciallyavailable. These include but are not limited to antibodies against theDYKDDDDK (SEQ ID NO:1) epitope, c-myc antibodies (available from Sigma,St. Louis), the HNK-1 carbohydrate epitope, the HA epitope, the HSVepitope, the His₄, His₅, and His₆ epitopes that are recognized by theHis epitope specific antibodies (see, e.g., Qiagen), and the like. Inaddition, vectors for epitope tagging proteins are commerciallyavailable. Thus, for example, the pCMV-Tag1 vector is an epitope taggingvector designed for gene expression in mammalian cells. A target geneinserted into the pCMV-Tag1 vector can be tagged with the FLAG® epitope(N-terminal, C-terminal or internal tagging), the c-myc epitope(C-terminal) or both the FLAG (N-terminal) and c-myc (C-terminal)epitopes.

“High-content screening” (HCS) in cell-based systems is a method thattypically uses living cells as to elucidate the workings of normal anddiseased cells. High-content screening technology is mainly based onautomated digital microscopy and, optionally, flow cytometry, typicallyin combination with IT-systems for the analysis and storage of the data.“High-content” or visual biology technology has two purposes, first toacquire spatially or temporally resolved information on an event andsecond to automatically quantify it. Spatially resolved instruments aretypically automated microscopes, and temporal resolution still requiressome form of fluorescence measurement in most cases. This means thatmany HCS instruments are (fluorescence) microscopes that are connectedto some form of image analysis package. These take care of all the stepsin taking fluorescent images of cells and provide rapid, automated andunbiased assessment of experiments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Amino acid sequences of VH domains of HCA-F1 (SEQ ID NO:2),HCA-F2 (SEQ ID NO:3), HCA-M1 (SEQ ID NO:4), and HCA-S1 (SEQ ID NO:5)antibodies and amino acid sequences of VL domains of HCA-F1 (SEQ IDNO:6), HCA-F2 (SEQ ID NO:7), HCA-M1 (SEQ ID NO:8), and HCA-S1 (SEQ IDNO:9) antibodies.

FIGS. 2A-2C. Outline of screening strategy and data from the first stepof the screening, i.e., phage binding to DU145 cells. FIG. 2A) Schematicof HCA screening to identify macropinocytosis-dependent antibodies. HCAinstruments allow automated high throughput detection of antibodycolocalization with a macropinocytosis marker. The starting materialsfor the screening are sublibraries generated previously by us fromLCM-based phage antibody library selection [1] that are enriched forinternalizing phage antibodies binding to tumor cells in situ. FIG. 2B)DU145 cells were incubated in 96-well plates with phage-containingsupernatants for 24 hours at 37° C. in complete DMEM/10% FBS. Nucleiwere stained with Hoechst 33342. Bound phages were immunolabeled withanti-Fd antibodies (green). Zoomed insert portrays software-based,automated cell analysis, measuring mean fluorescence intensities (MFI)of immunolabeled phages. Over 300 cells were quantified for each phageclone. FIG. 2C) Plot of MFI values of immunolabeled phage binding tocell for 1,439 phage clones. Red horizontal line represents MFI of˜250,000, the threshold for prioritizing clones for furtherinternalization analysis.

FIG. 3, panels A-E show colocalization of phage antibodies with themacropinocytosis marker ND70-TR. Panel A) Epifluorescent images of DU145cells that were incubated with phage-containing supernatants and 50μg/ml ND70-TR (red) for 24 h at 37° C. Cell-associated phage were thendetected by biotin-labeled anti-fd antibody followed bystreptavidin-AlexaFluor 488 (green). Colocalization results in coloroverlap (yellow). Panel B) To analyze colocalization, arbitrary lineswere drawn across cells and fluorescent intensities along the drawn linewere plotted for phages (green) and ND70-TR fluorescence (red).Co-variation of line intensity indicates colocalization. Representativeimages of two different phage antibodies with differing colocalizationpatterns are shown. Panel C) Pearson's correlation coefficient (PCC) wasquantified and averaged from >30 cells per phage conditions. Error barsdenote SEM for n=3; * and ** indicate P-values of <0.05 and <0.01,respectively, using two-tailed student's T-tests assuming unequalvariance. Scale bar denotes 20 μm. Panel D) Colocalization screening.DU145 cells were plated onto 96-well plates and incubated with phagesand ND70-TR (red) for 24 h at 37° C. Cells were immunolabeled againstbacteriophages (green) and nuclei were stained with Hoechst 33342(blue). Panel E) Mean PCC between immunolabeled phage and ND70-TR of 360phage clones, quantified from minimum of 300 cells per phage clone. PCCvalues were normalized to control phage clones that exhibited poorinternalization. Green horizontal line represents 200% of control, athreshold for further analysis.

FIG. 4, panels A-C show confocal analysis of phage antibodyinternalization by DU145 cells. Confocal Z-slices of DU145 cellsincubated with purified phage for Panel A) 24 h at 37° C. or Panel B) 8h at 37° C. in the presence of ND70-TR. Cells were immunolabeled againstphages (green), lysosomes (LAMP1, red), and nuclei (Hoechst 33342,blue). Scale bar: 20 μm. Panel C) Mean PCC of internalized phages andND70-TR. Over 30 cells were analyzed per phage antibody. ** denotestwo-tailed t-test P-values of <0.01. Error bars represent SEM for n=3.

FIG. 5, panels A-B, show internalization and colocalization analysis ofIgGs derived from scFvs. Panel A) DU145 cells co-incubated with threeIgGs with different internalization properties at 10 μg/ml and 50 μg/mlND70-TR (red) for 90 min at 37° C. Cells were immunolabeled against IgGusing anti-human Fc (green). Nuclei were stained with Hoechst 33342(blue). Single confocal Z-slice images are shown. Scale bar: 20 μm.Panel B) PCC analysis of colocalization of IgGs HCA-F1, HCA-M1, andHCA-S1 with ND70-TR using Z-slices crossing the entire cell,quantitating a minimum of 10 cells. ** and *** denote two-tailed t-testP-values of <0.01 and <0.001, respectively. Error bars represent SEM forn=3.

FIG. 6, panels A-D show kinetics of antibody internalization andsubcellular localization. DU145 cells were incubated with threedifferent IgGs (HCA-F1, HCA-M1, or HCA-S1) at 10 μg/ml for 15 min at 4°C. and then chased with complete DMEM/10% FBS for indicated timeperiods. Cells were then fixed, permeabilized and immunolabeled againsthuman IgG (green) and Panel A) early endosomes (EEA1, red) or Panel B)lysosomes (LAMP1, red). Nuclei were stained with Hoechst 33342 (blue).Scale bar: 20 μm. Pearson's correlation coefficients betweenimmunolabeled Panel C) EEA1 or Panel D) LAMP1 and immunolabeled IgG wereaveraged from a minimum of 30 cells. Error bars denote SEM of n=3.

FIG. 7, panels A-B show that macropinocytosis inhibitors preventinternalization of IgG HCA-F1. DU145 cells were pre-treated with 50μg/ml cytochalasin D, 7.5 μg/ml EIPA, or DMSO (control) for 30 min at37° C. followed by co-incubation with 10 μg/ml IgG HCA-F1 and ND70-TR(red) in the presence of cytochalasin D, EIPA, or DMSO in completeDMEM/10% FBS for 40 min at 37° C. Cells were then immunolabeled forhuman IgG (green). Nuclei were stained with Hoechst 33342 (blue). PanelA) Individual confocal Z-slices of representative cells. CytoD:cytochalasin D. Scale bar: 20 μm. Panel B) The percentage ofinternalized IgG HCA-F1 was quantitated by measuring the ratio ofinternalized, cytosolic IgG HCA-F1 fluorescence over total cell IgGHCA-F1 fluorescence, analyzing >15 cells over 3 independent experiments.CytoD: cytochalasin D. *** indicates P-value of <0.001 using two-tailedstudent's T-test assuming unequal variance. Error bars represent SEMwith n=3.

FIG. 8, panels A-C, show EphA2 identified as target antigen bound bymacropinocytosing antibody IgG HCA-F1. Panel A) Immunoprecipiation ofthe target antigen from surface-biotinylated Du145 whole cell lysatesusing scFv HCA-F1-Fc fusion immobilized onto a solid matrix. Theimmunoprecipitation product was run on SDS-PAGE and subjected to Westernblot analysis using streptavidin-HRP to locate the position of membraneproteins. The dominant band, denoted by “*”, represents the approximateregion from which the corresponding SDS-PAGE gel was extracted for massspectrometry analysis. Panel B) Binding to ectopically expressed EphA2.Chinese hamster ovarian (CHO) cells were co-transfected with pEGFP-N2(to label transfected cells) and pCMV6 expression constructs bearingeither human EphA2 or Lgr5 (control). Cells were then incubated with IgGHCA-F1, followed by immunolabeling using anti-human Fc AlexaFluor 647.Cells were gated for GFP expression and plotted for AlexaFluor 647fluorescence (FL4). Panel C) Plot of MFI values as analyzed by FACS. IgGHCA-F1 binds specifically to ectopically expressed EphA2, confirming thetarget identification.

FIG. 9, panels A-B show functional internalization assay using IgGHCA-F1-toxin conjugates. Panel A) FACS analysis showing EphA2-positive(DU145) and EphA2-negative (LNCaP, control) cells. IgG HCA-F1 wasincubated with the cells and binding was detected with anti-human Fc.MFI values are shown in the far right panel. Panel B) IgG HCA-F1 wasconjugated to saporin and incubated with target (DU145) and control(LNCaP) cells. Controls: toxin only and IgG HCA-F1 only. Cell viabilitywas measured 4 days later using the CCK-8 assay.

FIG. 10, panels A-B, shows patterns of cell-associated phage antibodies.Panel A) Various patterns of cell-associated phage antibodies. DU145cells were incubated with phage containing supernatants at 37° C. for 2h, washed, fixed, permeabilized, and phage detected by anti-fdantibodies (green). Nuclei were stained with Hoechst 33342. Scale bar:20 μm. Panel B) Summary of phage antibody patterns; n=13 unique phageclones. These patterns are not mutually exclusive as a monoclonal phageoften exhibits multiple patterns as indicated

FIG. 11 shows FACS analysis of phage antibody binding to DU145 cells.Bound phage were detected by biotin-labeled anti-fd antibody followed bystreptavidin-PE. MFI values are shown in the right panel.

FIG. 12 shows resistance to high-salt, low-pH glycine buffer washes bycell surface-bound phage antibodies. DU145 cells were first fixed andthen incubated with phage-containing supernatants, followed by washeswith a pH 2.8 buffer containing 500 mM NaCl and 100 mM glycine. Phageswere immunolabeled using biotin-labeled rabbit anti-fd antibodiesfollowed by streptavidin-cy3 (pseudo-colored as green). Nuclei werestained with Hoechst 33342. Scale bar: 20 μm.

FIG. 13 show results of quality control studies: IgGs derived from scFvbind to DU145 cells. FACS analysis of DU145 cells incubated with IgGHCA-F1, HCA-M1, and HCA-S1 at 10 μg/ml for 90 min at RT. Cell-bound IgGswere detected by anti-human Fc secondary antibody conjugated withAlexaFluor 647.

FIG. 14, panels A-B, shows that IgG HCA-F1 does not significantlycolocalize with caveolin or clathrin heavy chain. DU145 cells pulsedwith 10 μg/ml IgG HCA-F1 (green) in complete DMEM/10% FBS for 30 min at4° C. were chased with 37° C. DMEM/10% FBS and fixed at varying timepoints. Cells were immunolabeled for organelles (red) Panel A) caveolin(Cav2) and Panel B) clathrin heavy chain (CHC). Nuclei were stained withHoechst 33342. Single confocal Z-slices are shown. Scale bar: 20 μm.

FIG. 15 shows that ScFv-Fc HCA-F1 internalizes and colocalizes withearly endosomes and lysosomes in DU145 cells. DU-145 cells pulsed with10 μg/ml scFv-Fc HCA-F1 for 30 min at 4° C. were then chased with 37° C.pre-warmed media and fixed at varying time points. Cells wereimmunolabeled against scFv-Fc HCA-F1 (green) and intracellularorganelles (red), including early endosomes (EEA1) and lysosomes(LAMP1). Nuclei were stained with Hoechst 33342. Single confocalZ-slices are shown. Scale bar: 20 μm.

FIG. 16 shows MFI value plot showing IgG HCA-F1 binding patterns on apanel of cancer and non-cancer cell lines. Cells were incubated with 10μg/ml IgG HCAF1, washed and bound IgG detected with anti-human Fc.

FIG. 17, panels A-B, shows that IgG HCA-F1 preferentially internalizesinto cancer cell lines when compared to non-cancer cell lines. Panel A)Confocal Z-projection micrographs of various cells incubated with 10μg/ml IgG HCA-F1 and 20 μg/ml ND70-TR for 90 min at 37° C., followed byimmunolabeling against human Fc. Nuclei were stained with Hoechst 33342.The five cancer cell lines are on the left (MDA-MB-231, DU145, HeLa,A549, and A431) while the two non-cancer cell lines are on the right(293A and BPH1). Scale bar: 15 μm. Panel B) Average internalized IgGHCA-F1 integrated intensity/area for each cell line, measuring a minimumof 10 cells. Error bars denote SEM from n=3.

FIG. 18 illustrates potent tumor cell killing by a macropinocytosingantibody-drug conjugate (ADC).

DETAILED DESCRIPTION

In various embodiments, methods are provided for identifying andselecting antibodies that are internalized into cells via themacropinocytosis pathway. Additionally antibodies that are internalizedvia this pathway are provided as well as immunoconjugates comprisingsuch antibodies.

Methods of Identifying Antibodies Internalized by the MacropinocytosisPathway.

In various embodiments, methods of preparing antibodies that areinternalized into a cell by a macropinocytosis pathway are provided. Inone illustrative, but non-limiting embodiment, the method involvescontacting target cells with members of an antibody library and withmarker(s) for macropinocytosis; identifying internalized antibodies thatco-localize in the target cells with the marker(s) for macropinocytosis;and selecting those antibodies that co-localize with the marker(s) formacropinocytosis. In various embodiments, the members of an antibodylibrary are members of a phage display library or members of a yeastdisplay library. In certain embodiments the antibody library is anantibody library that is enriched for antibodies that bind to tumorcells and said enrichment is by laser capture microdissection (LCM) ofantibodies that bind to tumor cells (e.g., as described in Ruan et al.(2006) Mol. Cell Proteomics. 5: 2364-2373, and in U.S. Ser. No.12/724,282 and PCT/US2008/076704 which are incorporated herein byreference for the LCM enrichment methods described therein). In certainembodiments, the methods involve selecting internalized antibodies thatcolocalize with a lysosomal marker (e.g., LAMP1). As described hereinand illustrated in the examples, the method is facilitated by the use ofhigh content analysis (HCA) using digital microscopy and a dataacquisition system.

One illustrative, but non-limiting embodiment of the HCA-based strategythat used to identify antibodies capable of internalizing into tumorcells via macropinocytosis is outlined in FIG. 2A. An HCA platform wasdeveloped that allows quantitative measurement of colocalization betweenphage antibodies and a macropinocytic marker (e.g., ND70-TR,FITC-dextran, latex or glass beads, Lucifer yellow, etc.). To identifyclinically relevant macropinocytosing antibodies, we screened phageantibody libraries that we have generated previously by laser capturemicrodissection (LCM)-based selection, which are highly enriched forinternalizing antibodies that bind to prostate tumor cells in situresiding in the tumor tissue microenvironment [1]. More particularly,the HCA protocol was used to screen single chain variable fragment(scFv) phage antibody display libraries that were previously generatedby laser capture microdissection (LCM)-based selection on live tumorcells and tumor tissues, which are highly enriched for internalizingphage antibodies binding to prostate tumor cells in situ residing intheir tissue microenvironment [1], and identified antibodies that arecapable of efficient internalization via macropinocytosis.

Antibodies Internalized by the Macropinocytosis Pathway.

In certain embodiments antibodies that are internalized into cells bythe macropinocytosis pathway are provided. The antibodies wereidentified by selecting human antibody gene diversity libraries directlyon the surface of prostate cancer cells in vivo using lasermicrodissection methods as described above and in the examples.Antibodies were identified that specifically bind and enter prostatecancer cells, with little or no binding to control cells.

For the selection process, the antibodies in the library were expressedas single chain Fv (scFv) antibodies comprising a variable heavy (V_(H))region linked to a variable light (V_(L)) region by a peptide linker,although it will be recognized that using the antibody sequencepresented herein other forms of the antibodies can be provided.

Representative antibodies (e.g. V_(H) and V_(L) domains) are illustratedin Table 1 and FIG. 1.

TABLE 1 Amino acid sequences of VL and VH domains of HCA-F1 and HCA-F2antibodies internalized by the macropinocytosis pathway. HCA-M1 isinternalized at a moderate rate while HCA-S1 is slowly internalized incontrast to the macropinocytosing antibodies (HCA-F1 and HCA-F2). HCA-M1and HCA-S1 do not bind to EphA2, but they are useful for other applicationswhere non-internalizing antibodies are desired (such as bispecific mAbs forT cell capture etc.). Heavy chain Clone Frame 1 CDR1 Frame 2 CDR2Frame 3 CDR3 Frame 4 HCA-F1 QVQLQES SYSMN WVRQAPG YISS RFTISRD YRLPWGQGTTV SEQ ID GGGLVQP KGLEWVS SSST NAKNSLY DFWS TVSS NO: 2 GGSLRLS IYYALQMNSLR GYN CAASGFT DSVK AEDTAVY YGMD FS G YCAR v HCA-F2 QVQLVES SYAMSWVRQAPG AISG RFTISRD LSVE WGQGTLV SEQ ID GGGLVQP KGLEWVS SGS NSKNTLYWYGS TVSS NO: 3 GGSLRLS TYYA LQMNSLR GSYL CAASGFT DSVK AEDTAVY GY FS GYCAT HCA-M1 QVQLVES SYAMH WVRQAPG VISY RFTISRD APAY WGQGTLV SEQ IDGGGVVQP KGLEWVA DGSN NSKNTLY SYGP TVSS NO: 4 GRSLRLS KYYA LQMNSLR FDYCAASGFT DSVK AEDTAVY FS G YCAR HCA-S1 QVQLQES SYAMH WVRQAPG VISY RFTISRDFSSG WGQGTLV SEQ ID GGGLVQP KGLEWVA DGSN NSKNTLY WYYF TVSS NO: 5 GGSLRLSKYYA LQMNSLR DY CAASGFT DSVK AEDTAVY FS G YCAR Light chain Clone Frame 1CDR1 Frame 2 CDR2 Frame 3 CDR3 Frame 4 HCA-F1 QSVLTQP TGSSS WYQQLPG YGNSGVPDRFS QSYD FGGGTKL SEQ ID PSVSGAP NIGAG TAPKLLI NRPS GSKSGTS SSLS TVLNO: 6 GQRVTIS YDVH ASLAITG GHVV C LQAEDEA DYYC HCA-F2 NFMLTQD QGDSLWYQQKPG YGKN GIPDRFS NSRD FGGGTKV SEQ ID PAVSVAL RSYYA QAPVLVI NRPSGSSSGNT SSAN TVL NO: 7 GQTVRIT S ASLTITG HVV C AQAEDEA HYYC HCA-M1SSELTQD QGDSL WYQQKPG YGKN GIPDRFS HSRD FGGGTKV SEQ ID PAVSVAL RSYYAQAPVLVI NRPS GSSSGNT SSGT TVL NO: 8 GQTVRIT S ASLTITG HLRV C AQAEDEADYYC HCA-S1 DIQMTQS RASHD WYQQKPG YAAS GVPSRFS QQLG FGGGTKL SEQ IDPSFLSAS ISSYF KAPKPLI TLQS GSGSGTE SYPL EIK NO: 9 VGDRITI A FTLTISS T TCLQPEDFA TYYC

In certain embodiments, for single chain Fv antibodies the variableheavy (VH) region is coupled to the variable light (V_(L)) eitherdirectly, or more preferably by a peptide linker (e.g., (Gly₄Ser)₃, SEQID NO:10).

Using the sequence information provided in Table 1 and/or FIG. 1,antibodies macropinocytosing antibodies HCA-F1, and HCA-F2, or HCA-M1,and HCA-S1, or antibodies comprising one or more of the CDRs comprisingthese antibodies, or antibodies comprising the VH and/or VL domain(s) ofthese antibodies can readily be prepared using standard methods (e.g.chemical synthesis methods and/or recombinant expression methods) wellknown to those of skill in the art.

In addition, other “related” antibodies that are internalized by themacropinocytosis pathway can be identified by screening for antibodiesthat bind to the same epitope (e.g. that compete with the listedantibodies for binding to ephrin type-A receptor 2 (EphA2) and/or bymodification of the antibodies identified herein to produce libraries ofmodified antibody and then rescreening antibodies in the library forimproved internalization by the macropinocytosis pathway, and/or byscreening of various libraries on cancer cells, e.g., as illustrated inExample 1.

A) Chemical Synthesis.

Using the sequence information provided herein, the antibodiesinternalized by the macropinocytosis pathway (e.g., HCA-F1, HCA-F2,etc.), or variants thereof, can be chemically synthesized using wellknown methods of peptide synthesis. Solid phase synthesis in which theC-terminal amino acid of the sequence is attached to an insolublesupport followed by sequential addition of the remaining amino acids inthe sequence is one preferred method for the chemical synthesis ofsingle chain antibodies. Techniques for solid phase synthesis aredescribed by Barany and Merrifield, Solid Phase Peptide Synthesis; pp.3-284 in The Peptides: Analysis, Synthesis, Biology. Vol. 2: SpecialMethods in Peptide Synthesis, Part A., Merrifield et al. (1963) J. Am.Chem. Soc., 85: 2149-2156, and Stewart et al. (1984) Solid Phase PeptideSynthesis, 2nd ed. Pierce Chem. Co., Rockford, Ill.

B) Recombinant Expression of Prostate Cancer-Specific Antibodies.

In certain preferred embodiments, the antibodies internalized by themacropinocytosis pathway (e.g., HCA-F1, HCA-F2, etc.), or variantsthereof, are prepared using standard techniques well known to those ofskill in the art. Using the sequence information provided herein,nucleic acids encoding the desired antibody can be chemicallysynthesized according to a number of standard methods known to those ofskill in the art. Oligonucleotide synthesis, is preferably carried outon commercially available solid phase oligonucleotide synthesis machines(Needham-VanDevanter et al. (1984) Nucleic Acids Res. 12: 6159-6168) ormanually synthesized using the solid phase phosphoramidite triestermethod described by Beaucage et. al. (1981) Tetrahedron Letts. 22(20):1859-1862. Alternatively, nucleic acids encoding the antibody can beamplified and/or cloned according to standard methods.

Molecular cloning techniques to achieve these ends are known in the art.A wide variety of cloning and in vitro amplification methods aresuitable for the construction of recombinant nucleic acids. Examples ofthese techniques and instructions sufficient to direct persons of skillthrough many cloning exercises are found in Berger and Kimmel, Guide toMolecular Cloning Techniques, Methods in Enzymology volume 152 AcademicPress, Inc., San Diego, Calif. (Berger); Sambrook et al. (1989)Molecular Cloning—A Laboratory Manual (2nd ed.) Vol. 1-3, Cold SpringHarbor Laboratory, Cold Spring Harbor Press, NY, (Sambrook); and CurrentProtocols in Molecular Biology, F. M. Ausubel et al., eds., CurrentProtocols, a joint venture between Greene Publishing Associates, Inc.and John Wiley & Sons, Inc., (1994 Supplement) (Ausubel). Methods ofproducing recombinant immunoglobulins are also known in the art. See,Cabilly, U.S. Pat. No. 4,816,567; and Queen et al. (1989) Proc. NatlAcad. Sci. USA 86: 10029-10033. In addition, detailed protocols for theexpression of antibodies are also provided by Liu et al. (2004) CancerRes. 64: 704-710, Poul et al. (2000) J. Mol. Biol. 301: 1149-1161, andthe like.

C) Identification of Other Antibodies Binding the Same Target asAntibodies HCA-F1, HCA-F2, HCA-M1, and/or HCA-S1.

Having identified useful antibodies internalized by the macropinocytosispathway (e.g., HCA-F1, HCA-F2), other “related” antibodies internalizedby the macropinocytosis pathway can be identified by screening forantibodies that cross-react with the identified antibodies, e.g., atephrin type-A receptor 2 (EphA2) or at the epitope of EphA2 bound byHCA-F1, HCA-F2, HCA-M1, and/or HCA-S1 and/or with an idiotypic antibodyraised against HCA-F1, HCA-F2, HCA-M1, and/or HCA-S1 antibody.

1) Cross-Reactivity with Anti-Idiotypic Antibodies.

The idiotype represents the highly variable antigen-binding site of anantibody and is itself immunogenic. During the generation of anantibody-mediated immune response, an individual will develop antibodiesto the antigen as well as anti-idiotype antibodies, whose immunogenicbinding site (idiotype) mimics the antigen.

Anti-idiotypic antibodies can be raised against the variable regions ofthe antibodies identified herein using standard methods well known tothose of skill in the art. Briefly, anti-idiotype antibodies can be madeby injecting the antibodies of this invention, or fragments thereof(e.g., CDRs) into an animal thereby eliciting antisera against variousantigenic determinants on the antibody, including determinants in theidiotypic region.

Methods for the production of anti-analyte antibodies are well known inthe art. Large molecular weight antigens (greater than approx. 5000Daltons) can be injected directly into animals, whereas small molecularweight compounds (less than approx. 5000 Daltons) are preferably coupledto a high molecular weight immunogenic carrier, usually a protein, torender them immunogenic. The antibodies produced in response toimmunization can be utilized as serum, ascites fluid, an immunoglobulin(Ig) fraction, an IgG fraction, or as affinity-purified monospecificmaterial.

Polyclonal anti-idiotype antibodies can be prepared by immunizing ananimal with the antibodies of this invention prepared as describedabove. In general, it is desirable to immunize an animal which isspecies and allotype-matched with the animal from which the antibody(e.g. phage-display library) was derived. This minimizes the productionof antibodies directed against non-idiotypic determinants. The antiserumso obtained is then usually absorbed extensively against normal serumfrom the same species from which the phage-display library was derived,thereby eliminating antibodies directed against non-idiotypicdeterminants. Absorption can be accomplished by passing antiserum over agel formed by crosslinking normal (nonimmune) serum proteins withglutaraldehyde. Antibodies with anti-idiotypic specificity will passdirectly through the gel, while those having specificity fornon-idiotypic determinants will bind to the gel. Immobilizing nonimmuneserum proteins on an insoluble polysaccharide support (e.g., sepharose)also provides a suitable matrix for absorption.

Monoclonal anti-idiotype antibodies can be produced using the method ofKohler et al. (1975) Nature 256: 495. In particular, monoclonalanti-idiotype antibodies can be prepared using hybridoma technologywhich comprises fusing (1) spleen cells from a mouse immunized with theantigen or hapten-carrier conjugate of interest (i.e., the antibodies orthis invention or subsequences thereof) to (2) a mouse myeloma cell linewhich has been selected for resistance to a drug (e.g., 8-azaguanine).In general, it is desirable to use a myeloma cell line which does notsecrete an immunoglobulin. Several such lines are known in the art. Onegenerally preferred cell line is P3X63Ag8.653. This cell line is ondeposit at the American Type Culture Collection as CRL-1580.

Fusion can be carried out in the presence of polyethylene glycolaccording to established methods (see, e.g., Monoclonal Antibodies, R.Kennett, J. McKearn & K. Bechtol, eds. N.Y., Plenum Press, 1980, andCurrent Topics in Microbiology & Immunology, Vol. 81, F. Melchers, M.Potter & N. L. Warner, eds., N.Y., Springer-Verlag, 1978). The resultantmixture of fused and unfused cells is plated out inhypoxanthine-aminopterin-thymidine (HAT) selective medium. Under theseconditions, only hybrid cells will grow.

When sufficient cell growth has occurred, (typically 10-14 dayspost-fusion), the culture medium is harvested and screened for thepresence of monoclonal idiotypic, anti-analyte antibody by any one of anumber of methods which include solid phase RIA and enzyme-linkedimmunosorbent assay. Cells from culture wells containing antibody of thedesired specificity are then expanded and recloned. Cells from thosecultures that remain positive for the antibody of interest are thenusually passed as ascites tumors in susceptible, histocompatible,pristane-primed mice.

Ascites fluid is harvested by tapping the peritoneal cavity, retestedfor antibody, and purified as described above. If a nonsecreting myelomaline is used in the fusion, affinity purification of the monoclonalantibody is not usually necessary since the antibody is alreadyhomogeneous with respect to its antigen-binding characteristics. Allthat is necessary is to isolate it from contaminating proteins inascites, i.e., to produce an immunoglobulin fraction.

Alternatively, the hybrid cell lines of interest can be grown inserum-free tissue culture and the antibody harvested from the culturemedium. In general, this is a less desirable method of obtaining largequantities of antibody because the yield is low. It is also possible topass the cells intravenously in mice and to harvest the antibody fromserum. This method is generally not preferred because of the smallquantity of serum which can be obtained per bleed and because of theneed for extensive purification from other serum components. However,some hybridomas will not grow as ascites tumors and therefore one ofthese alternative methods of obtaining antibody must be used.

2) Cross-Reactivity with the HCA-F1, HCA-F2, HCA-M1, and/or HCA-S1Antibodies.

In another approach, other antibodies internalized by themacropinocytosis pathway can be identified by the fact that they bindephrin type-A receptor 2 (EphA2) or at the epitope of EphA2 bound by“prototypic” antibodies described herein (e.g., HCA-F1, HCA-F2, etc.).

Methods of determining antibody cross-reactivity are well known to thoseof skill in the art. Generally the epitope bound by the prototypicantibodies of this invention is determined e.g. by epitope mappingtechniques. Methods of epitope mapping are well known to those of skillin the art (see, e.g., Reyes et al. (1992) Hepatitis E Virus (HEV):Epitope Mapping and Detection of Strain Variation, Elsevier SciencePublisher Shikata et al. eds., Chapter 43:237-245; Li et al. (1993)Nature 363: 85-88). Epitope mapping can be performed using Novatopesystem, a kit for which is commercially available from Novagen, Inc.

In certain embodiments, cross-reactive antibodies internalized by themacropinocytosis pathway show at least 60%, preferably 80%, morepreferably 90%, and most preferably at least 95% or at least 99%cross-reactivity with one or more of HCA-F1, HCA-F2, HCA-M1, and/orHCA-S1.

D) Phage Display Methods to Select Other “Related” AntibodiesInternalized by the Macropinocytosis Pathway.

1) Chain Shuffling Methods.

One approach to creating modified single-chain antibody (scFv) generepertoires has been to replace the original V_(H) or V_(L) gene with arepertoire of V-genes to create new partners (chain shuffling) (Clacksonet al. (1991) Nature. 352: 624-628). Using chain shuffling and phagedisplay (or yeast display) as well as the screening/selection methoddescribed above for identifying antibodies internalized by themacropinocytosis pathway, other suitable internalizing antibodies canreadily be identified.

Thus, for example a mutant scFv gene repertoire can be createdcontaining a V_(H) gene of the prototypic antibodies (e.g. as shown inTable 1 and/or FIG. 1) antibody and a human V_(L) gene repertoire (lightchain shuffling). The scFv gene repertoire can be cloned into a phagedisplay vector, e.g., pHEN-1 (Hoogenboom et al. (1991) Nucleic AcidsRes., 19: 4133-4137) or other vectors, and after transformation alibrary of transformants is obtained.

Similarly, for heavy chain shuffling, the antibodies internalized by themacropinocytosis pathway (e.g., HCA-F1, HCA-F2, HCA-M1, and/or HCA-S1,etc.) V_(H) CDR1 and/or CDR2, and/or CDR3 and light chain (see, e.g.,Table 1) are cloned into a vector containing a human V_(H) generepertoire to create a phage antibody library transformants. Fordetailed descriptions of chain shuffling to increase antibody affinitysee, e.g., Schier et al. (1996) J. Mol. Biol., 255: 28-43, and the like.

2) Site-Directed Mutagenesis to Improve Binding Affinity.

The majority of antigen contacting amino acid side chains are typicallylocated in the complementarity determining regions (CDRs), three in theV_(H) (CDR1, CDR2, and CDR3) and three in the V_(L) (CDR1, CDR2, andCDR3) (Chothia et al. (1987) J. Mol. Biol., 196: 901-917; Chothia et al.(1986) Science, 233: 755-8; Nhan et al. (1991) J. Mol. Biol., 217:133-151). These residues contribute the majority of binding energeticsresponsible for antibody affinity for antigen. In other molecules,mutating amino acids which contact ligand has been shown to be aneffective means of increasing the affinity of one protein molecule forits binding partner (Lowman et al. (1993) J. Mol. Biol., 234: 564-578;Wells (1990) Biochemistry, 29: 8509-8516). Site-directed mutagenesis ofCDRs of the antibodies described herein and screening forinternalization via the macropinocytosis pathway as described herein canproduce additional suitable antibodies.

3) CDR Randomization to Produce Higher Affinity Human scFv.

In an extension of simple site-directed mutagenesis, mutant antibodylibraries can be created where partial or entire CDRs are randomized(V_(L) CDR1 CDR2 and/or CDR3 and/or V_(H) CDR1, CDR2 and/or CDR3). Inone embodiment, each CDR is randomized in a separate library, using aknown antibody (e.g., HCA-F1, HCA-F2, HCA-M1, and/or HCA-S1) as atemplate. The CDR sequences of the best internalizing mutants from eachCDR library can be combined to obtain additional antibodies.

V_(H) CDR3 often occupies the center of the binding pocket, and thusmutations in this region are likely to result in an increase in affinity(Clackson et al. (1995) Science, 267: 383-386). In one embodiment, fourV_(H) CDR3 residues are randomized at a time (see, e.g., Schier et al.(1996) Gene, 169: 147-155; Schier and Marks (1996) Human Antibodies andHybridomas. 7: 97-105, 1996; and Schier et al. (1996) J. Mol. Biol. 263:551-567).

E) Creation of Other Antibody Forms.

Using the known and/or identified sequences (e.g. V_(H) and/or V_(L)sequences) of the HCA-F1, HCA-F2, HCA-M1, and/or HCA-S1 antibodies shownin Table 1 other antibody forms can readily be created. Such formsinclude, but are not limited to multivalent antibodies, full antibodies(e.g., IgG, IgA, IgM), scFv, (scFv′)₂, Fab, (Fab)₂, chimeric antibodies,and the like.

1) Creation of Homodimers.

For example, to create (scFv′)₂ antibodies, two scFV antibodiesinternalized by the macropinocytosis pathway are joined, either througha linker (e.g., a carbon linker, a peptide, etc.) or through a disulfidebond between, for example, two cysteins. Thus, for example, to createdisulfide linked scFv, a cysteine residue can be introduced by sitedirected mutagenesis at the carboxy-terminus of the antibodies describedherein.

An scFv can be expressed from this construct, purified by IMAC, andanalyzed by gel filtration. To produce (scFv′)₂ dimers, the cysteine isreduced by incubation with 1 mM 3-mercaptoethanol, and half of the scFvblocked by the addition of DTNB. Blocked and unblocked scFvs areincubated together to form (scFv′)₂ and the resulting material can beanalyzed by gel filtration. The affinity of the resulting dimmer can bedetermined using standard methods, e.g. by BIAcore.

In one illustrative embodiment, the (scFv′)₂ dimer is created by joiningthe scFv′ fragments through a linker, more preferably through a peptidelinker. This can be accomplished by a wide variety of means well knownto those of skill in the art. For example, one preferred approach isdescribed by Holliger et al. (1993) Proc. Natl. Acad. Sci. USA, 90:6444-6448 (see also WO 94/13804).

It is noted that using the V_(H) and/or V_(L) sequences provided hereinFabs and (Fab′)₂ dimers can also readily be prepared. Fab is a lightchain joined to V_(H)—C_(H)1 by a disulfide bond and can readily becreated using standard methods known to those of skill in the art. TheF(ab)′₂ can be produced by dimerizing the Fab, e.g. as described abovefor the (scFv′)₂ dimer.

2) Chimeric Antibodies.

The antibodies contemplated herein also include “chimeric” antibodies inwhich a portion of the heavy and/or light chain is identical with orhomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is identical with orhomologous to corresponding sequences in antibodies derived from anotherspecies or belonging to another antibody class or subclass, as well asfragments of such antibodies, so long as they exhibit the desiredbiological activity (see, e.g., U.S. Pat. No. 4,816,567; Morrison et al.(1984) Proc. Natl. Acad. Sci. 81: 6851-6855, etc.).

While the prototypic antibodies provided herein are fully humanantibodies, chimeric antibodies are contemplated, particularly when suchantibodies are to be used in species other than humans (e.g., inveterinary applications). Chimeric antibodies are antibodies comprisinga portions from two different species (e.g. a human and non-humanportion). Typically, the antigen combining region (or variable region)of a chimeric antibody is derived from a one species source and theconstant region of the chimeric antibody (which confers biologicaleffector function to the immunoglobulin) is derived from another source.A large number of methods of generating chimeric antibodies are wellknown to those of skill in the art (see, e.g., U.S. Pat. Nos. 5,502,167,5,500,362, 5,491,088, 5,482,856, 5,472,693, 5,354,847, 5,292,867,5,231,026, 5,204,244, 5,202,238, 5,169,939, 5,081,235, 5,075,431, and4,975,369, and PCT application WO 91/0996).

In general, the procedures used to produce chimeric antibodies consistof the following steps (the order of some steps may be interchanged):(a) identifying and cloning the correct gene segment encoding theantigen binding portion of the antibody molecule; this gene segment(known as the VDJ, variable, diversity and joining regions for heavychains or VJ, variable, joining regions for light chains, or simply asthe V or variable region or V_(H) and V_(L) regions) may be in eitherthe cDNA or genomic form; (b) cloning the gene segments encoding thehuman constant region or desired part thereof; (c) ligating the variableregion to the constant region so that the complete chimeric antibody isencoded in a transcribable and translatable form; (d) ligating thisconstruct into a vector containing a selectable marker and gene controlregions such as promoters, enhancers and poly(A) addition signals; (e)amplifying this construct in a host cell (e.g., bacteria); (f)introducing the DNA into eukaryotic cells (transfection) most oftenmammalian lymphocytes; and culturing the host cell under conditionssuitable for expression of the chimeric antibody.

Antibodies of several distinct antigen binding specificities have beenmanipulated by these protocols to produce chimeric proteins (e.g.,anti-TNP: Boulianne et al. (1984) Nature, 312: 643; and anti-tumorantigens: Sahagan et al. (1986) J. Immunol., 137: 1066). Likewiseseveral different effector functions have been achieved by linking newsequences to those encoding the antigen binding region. Some of theseinclude enzymes (Neuberger et al. (1984) Nature 312: 604),immunoglobulin constant regions from another species and constantregions of another immunoglobulin chain (Sharon et al. (1984) Nature309: 364; Tan et al., (1985) J. Immunol. 135: 3565-3567).

In certain embodiments, a recombinant DNA vector is used to transfect acell line to produce a cell that expresses antibodies internalized bythe macropinocytosis pathway. The novel recombinant DNA vector containsa “replacement gene” to replace all or a portion of the gene encodingthe immunoglobulin constant region in the cell line (e.g., a replacementgene may encode all or a portion of a constant region of a humanimmunoglobulin, a specific immunoglobulin class, or an enzyme, a toxin,a biologically active peptide, a growth factor, inhibitor, or a linkerpeptide to facilitate conjugation to a drug, toxin, or other molecule,etc.), and a “target sequence” that allows for targeted homologousrecombination with immunoglobulin sequences within the antibodyproducing cell.

In another embodiment, a recombinant DNA vector is used to transfect acell line that produces an antibody having a desired effector function,(e.g., a constant region of a human immunoglobulin) in which case, thereplacement gene contained in the recombinant vector may encode all or aportion of a region of an antibodies internalized by themacropinocytosis pathway and the target sequence contained in therecombinant vector allows for homologous recombination and targeted genemodification within the antibody producing cell. In either embodiment,when only a portion of the variable or constant region is replaced, theresulting chimeric antibody can define the same antigen and/or have thesame effector function yet be altered or improved so that the chimericantibody may demonstrate a greater antigen specificity, greater affinitybinding constant, increased effector function, or increased secretionand production by the transfected antibody producing cell line, etc.

Regardless of the embodiment practiced, the processes of selection forintegrated DNA (via a selectable marker), screening for chimericantibody production, and cell cloning, can be used to obtain a clone ofcells producing the chimeric antibody.

Thus, a piece of DNA that encodes a modification for a monoclonalantibody can be targeted directly to the site of the expressedimmunoglobulin gene within a B-cell or hybridoma cell line. DNAconstructs for any particular modification can be made to alter theprotein product of any monoclonal cell line or hybridoma. The level ofexpression of chimeric antibody should be higher when the gene is at itsnatural chromosomal location rather than at a random position. Detailedmethods for preparation of chimeric (humanized) antibodies can be foundin U.S. Pat. No. 5,482,856.

3) Intact Human Antibodies.

In another embodiment, this invention provides for intact, fully humanantibodies internalized by the macropinocytosis pathway. Such antibodiescan readily be produced in a manner analogous to making chimeric humanantibodies. In this instance, instead of using a recognition functionderived, e.g. from a murine, the fully human recognition function (e.g.,VH and V_(L)) of the antibodies described herein is utilized.

4) Diabodies.

In certain embodiments, diabodies comprising one or more of the V_(H)and V_(L) domains described herein are contemplated. The term“diabodies” refers to antibody fragments typically having twoantigen-binding sites. The fragments typically comprise a heavy chainvariable domain (V_(H)) connected to a light chain variable domain(V_(L)) in the same polypeptide chain (V_(H)-V_(L)). By using a linkerthat is too short to allow pairing between the two domains on the samechain, the domains are forced to pair with the complementary domains ofanother chain and create two antigen-binding sites. Diabodies aredescribed more fully in, for example, EP 404,097; WO 93/11161, andHolliger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448.

5) Unibodies.

In certain embodiments using the sequence information provided herein,the antibodies described herein can be constructed as unibodies. UniBodyantibody technology produces a stable, smaller antibody format with ananticipated longer therapeutic window than certain small antibodyformats. In certain embodiments unibodies are produced from IgG4antibodies by eliminating the hinge region of the antibody. Unlike thefull size IgG4 antibody, the half molecule fragment is very stable andis termed a uniBody. Halving the IgG4 molecule leaves only one area onthe UniBody that can bind to a target. Methods of producing unibodiesare described in detail in PCT Publication WO2007/059782, which isincorporated herein by reference in its entirety (see, also, Kolfschotenet al. (2007) Science 317: 1554-1557).

6) Affibodies.

In certain embodiments the sequence information provided herein is usedto construct affibody molecules are internalized by the macropinocytosispathway. Affibody molecules are class of affinity proteins based on a58-amino acid residue protein domain, derived from one of theIgG-binding domains of staphylococcal protein A. This three helix bundledomain has been used as a scaffold for the construction of combinatorialphagemid libraries, from which affibody variants that target the desiredmolecules can be selected using phage display technology (see, e.g.,Nord et al. (1997) Nat. Biotechnol. 15: 772-777; Ronmark et al. (2002)Eur. J. Biochem., 269: 2647-2655.). Details of Affibodies and methods ofproduction are known to those of skill (see, e.g., U.S. Pat. No.5,831,012 which is incorporated herein by reference in its entirety).

It will be recognized that the antibodies described above can beprovided as whole intact antibodies (e.g., IgG), antibody fragments, orsingle chain antibodies, using methods well known to those of skill inthe art. In addition, while the antibody can be from essentially anymammalian species, to reduce immunogenicity, it is desirable to use anantibody that is of the species in which the antibody and/or chimericmoiety is to be used. In other words, for use in a human, it isdesirable to use a human, humanized, or chimeric human antibody.

immunoconjugates Comprising Antibodies that are Internalized by theMacropinocytosis Pathway

The antibodies described herein that are internalized via amacropinocytosis pathway (e.g., HCA-F1, HCA-F2, etc.) can be used aloneas therapeutics (e.g., to inhibit growth and/or proliferation of aprostate cancer cell) or they can be coupled to an effector formingimmunoconjugates that provide efficient and specific delivery of theeffector (e.g. cytotoxins, labels, radionuclides, ligands, antibodies,drugs, liposomes, nanoparticles, viral particles, cytokines, and thelike) into cancer cells, particularly cancer cells where themacropinocytosis pathway is upregulated (e.g., ras-transformed cancercells).

Immunoconjugates can be formed by conjugating the antibodies or antigenbinding portions thereof described herein to an effector (e.g., adetectable label, another therapeutic agent, etc.). Suitable agentsinclude, for example, a cytotoxic or cytostatic agent (e.g., achemotherapeutic agent), a toxin (e.g. an enzymatically active toxin ofbacterial, fungal, plant or animal origin, or fragments thereof), and/ora radioactive isotope (i.e., a radioconjugate).

In certain embodiments, the effector comprises a detectable label.Suitable detectable labels include, but are not limited to radio-opaquelabels, nanoparticles, PET labels, MRI labels, radioactive labels, andthe like. Among the radionuclides and useful in various embodiments ofthe present invention, gamma-emitters, positron-emitters, x-ray emittersand fluorescence-emitters are suitable for localization, diagnosisand/or staging, and/or therapy, while beta and alpha-emitters andelectron and neutron-capturing agents, such as boron and uranium, alsocan be used for therapy.

The detectable labels can be used in conjunction with an externaldetector and/or an internal detector and provide a means of effectivelylocalizing and/or visualizing prostate cancer cells. Suchdetection/visualization can be useful in various contexts including, butnot limited to pre-operative and intraoperative settings. Thus, incertain embodiment this invention relates to a method ofintraoperatively detecting and prostate cancers in the body of a mammal.These methods typically involve administering to the mammal acomposition comprising, in a quantity sufficient for detection by adetector (e.g. a gamma detecting probe), an prostate cancer specificantibody labeled with a detectable label (e.g. antibodies of thisinvention labeled with a radioisotope, e.g. ¹⁶¹Tb, ¹²³I, ¹²⁵I, and thelike), and, after allowing the active substance to be taken up by thetarget tissue, and preferably after blood clearance of the label,subjecting the mammal to a radioimmunodetection technique in therelevant area of the body, e.g. by using a gamma detecting probe.

In certain embodiments the label-bound antibody can be used in thetechnique of radioguided surgery, wherein relevant tissues in the bodyof a subject can be detected and located intraoperatively by means of adetector, e.g. a gamma detecting probe. The surgeon can,intraoperatively, use this probe to find the tissues in which uptake ofthe compound labeled with a radioisotope, that is, e.g. a low-energygamma photon emitter, has taken place. In certain embodiments suchmethods are particularly useful in localizing and removing secondarycancers produced by metastatic cells from a primary tumor.

In addition to detectable labels, certain preferred effectors include,but are not limited to cytotoxins (e.g. Pseudomonas exotoxin, ricin,abrin, Diphtheria toxin, and the like), or cytotoxic drugs or prodrugs,in which case the chimeric molecule may act as a potent cell-killingagent specifically targeting the cytotoxin to prostate cancer cells.

In still other embodiments, the effector can include a liposomeencapsulating a drug (e.g. an anti-cancer drug such as abraxane,doxorubicin, pamidronate disodium, anastrozole, exemestane,cyclophosphamide, epirubicin, toremifene, letrozole, trastuzumab,megestroltamoxifen, paclitaxel, docetaxel, capecitabine, goserelinacetate, zoledronic acid, vinblastine, etc.), an antigen that stimulatesrecognition of the bound cell by components of the immune system, anantibody that specifically binds immune system components and directsthem to the prostate cancer, and the like.

Illustrative Effectors.

Imaging Compositions.

In certain embodiments, the macropinocytosis pathway internalizingantibodies can be used to direct detectable labels to and into a tumorsite. This can facilitate tumor detection and/or localization. It can beeffective for detecting primary tumors, or, in certain embodiments,secondary tumors produced by, e.g., prostate metastatic cells. Incertain embodiments, the effector component of the immunoconjugatecomprises a “radio-opaque” label, e.g. a label that can be easilyvisualized using x-rays. Radio-opaque materials are well known to thoseof skill in the art. The most common radio-opaque materials includeiodide, bromide or barium salts. Other radiopaque materials are alsoknown and include, but are not limited to, organic bismuth derivatives(see, e.g., U.S. Pat. No. 5,939,045), radio-opaque polyurethanes (see,e.g., U.S. Pat. No. 5,346,981), organobismuth composites (see, e.g.,U.S. Pat. No. 5,256,334), radio-opaque barium polymer complexes (see,e.g., U.S. Pat. No. 4,866,132), and the like.

The macropinocytosis pathway internalizing antibodies described hereincan be coupled directly to the radio-opaque moiety or they can beattached to a “package” (e.g., a chelate, a liposome, a polymermicrobead, a nanoparticle, etc.) carrying, containing, or comprising theradio-opaque material, e.g., as described below.

In addition to radio-opaque labels, other labels are also suitable foruse. Detectable labels suitable for use in immunoconjugates include anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Useful labelsinclude, but are not limited to radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C,or ³²P), PET labels, MRI labels, radio-opaque labels, and the like.

In certain embodiments, suitable radiolabels include, but are notlimited to, ⁹⁹TC, ²⁰³Pb, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ¹¹¹In, ^(113m)In, ⁹⁷Ru, ⁶²Cu,641Cu, ⁵²Fe, ^(52m)Mu, ⁵¹Cr, ¹⁸⁶Re, ¹⁸⁸Re, ⁷⁷As, ⁹⁰Y, ⁶⁷Cu, ¹⁶⁹Er,¹²¹Sn, ¹²⁷Te, ¹⁴²Pr, ¹⁴³Pr, ¹⁹⁸Au, ¹⁹⁹Au, ¹⁶¹Tb, ¹⁰⁹Pd, ¹⁶⁵Dy, ¹⁴⁹Pm,¹⁵¹Pm, ¹⁵³Sm, ¹⁵⁷Gd, ¹⁵⁹Gd, ¹⁶⁶Ho, ¹⁷²Tm, ¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁷⁷Lu, ¹⁰⁵Rh,and ¹¹¹Ag.

Means of detecting such labels are well known to those of skill in theart. Thus, for example, certain radiolabels may be detected usingphotographic film, scintillation detectors, PET imaging, MRI, and thelike. Fluorescent markers can be detected using a photodetector todetect emitted illumination. Enzymatic labels are typically detected byproviding the enzyme with a substrate and detecting the reaction productproduced by the action of the enzyme on the substrate, and colorimetriclabels are detected by simply visualizing the colored label.

Radiosensitizers.

In certain embodiments, the effector can comprise a radiosensitizer thatenhances the cytotoxic effect of ionizing radiation (e.g., such as mightbe produced by ⁶⁰Co or an x-ray source) on a cell. Numerousradiosensitizing agents are known and include, but are not limited tobenzoporphyrin derivative compounds (see, e.g., U.S. Pat. No.5,945,439), 1,2,4-benzotriazine oxides (see, e.g., U.S. Pat. No.5,849,738), compounds containing certain diamines (see, e.g., U.S. Pat.No. 5,700,825), BCNT (see, e.g., U.S. Pat. No. 5,872,107),radiosensitizing nitrobenzoic acid amide derivatives (see, e.g., U.S.Pat. No. 4,474,814), various heterocyclic derivatives (see, e.g., U.S.Pat. No. 5,064,849), platinum complexes (see, e.g., U.S. Pat. No.4,921,963), and the like.

Alpha Emitters.

In certain embodiments, the effector can include an alpha emitter, i.e.a radioactive isotope that emits alpha particles. Alpha-emitters haverecently been shown to be effective in the treatment of cancer (see,e.g., McDevitt et al. (2001) Science 294:1537-1540; Ballangrud et al.(2001) Cancer Res. 61: 2008-2014; Borchardt et al. (2003) Cancer Res.63: 5084-50). Suitable alpha emitters include, but are not limited toBi, ²¹³Bi, ²¹¹At, and the like.

Chelates

Many of the pharmaceuticals and/or radiolabels described herein can beprovided as a chelate. The chelating molecule is typically coupled to amolecule (e.g. biotin, avidin, streptavidin, etc.) that specificallybinds an epitope tag attached to a macropinocytosis pathwayinternalizing antibody described herein.

Chelating groups are well known to those of skill in the art. In certainembodiments, chelating groups are derived from ethylene diaminetetra-acetic acid (EDTA), diethylene triamine penta-acetic acid (DTPA),cyclohexyl 1,2-diamine tetra-acetic acid (CDTA),ethyleneglycol-O,O′-bis(2-aminoethyl)-N,N,N′,N′-tetra-acetic acid(EGTA), N,N-bis(hydroxybenzyl)-ethylenediamine-N,N′-diacetic acid(HBED), triethylene tetramine hexa-acetic acid (TTHA),1,4,7,10-tetraazacyclododecane-N,N′-,N″,N′″-tetra-acetic acid (DOTA),hydroxyethyldiamine triacetic acid (HEDTA),1,4,8,11-tetra-azacyclotetradecane-N,N′,N″,N′″-tetra-acetic acid (TETA),substituted DTPA, substituted EDTA, and the like.

Examples of certain preferred chelators include unsubstituted or,substituted 2-iminothiolanes and 2-iminothiacyclohexanes, in particular2-imino-4-mercaptomethylthiolane.

One chelating agent, 1,4,7,10-tetraazacyclododecane-N, N, N″,N′″-tetraacetic acid (DOTA), is of particular interest because of itsability to chelate a number of diagnostically and therapeuticallyimportant metals, such as radionuclides and radiolabels.

Conjugates of DOTA and proteins such as antibodies have been described.For example, U.S. Pat. No. 5,428,156 teaches a method for conjugatingDOTA to antibodies and antibody fragments. To make these conjugates, onecarboxylic acid group of DOTA is converted to an active ester which canreact with an amine or sulfhydryl group on the antibody or antibodyfragment. Lewis et al. (1994) Bioconjugate Chem. 5: 565-576, describes asimilar method wherein one carboxyl group of DOTA is converted to anactive ester, and the activated DOTA is mixed with an antibody, linkingthe antibody to DOTA via the epsilon-amino group of a lysine residue ofthe antibody, thereby converting one carboxyl group of DOTA to an amidemoiety.

In certain embodiments the chelating agent can be coupled, directly orthrough a linker, to an epitope tag or to a moiety that binds an epitopetag. Conjugates of DOTA and biotin have been described (see, e.g., Su(1995) J. Nucl. Med., 36 (5 Suppl):154P, which discloses the linkage ofDOTA to biotin via available amino side chain biotin derivatives such asDOTA-LC-biotin or DOTA-benzyl-4-(6-amino-caproamide)-biotin). Yau etal., WO 95/15335, disclose a method of producing nitro-benzyl-DOTAcompounds that can be conjugated to biotin. The method comprises acyclization reaction via transient projection of a hydroxy group;tosylation of an amine; deprotection of the transiently protectedhydroxy group; tosylation of the deprotected hydroxy group; andintramolecular tosylate cyclization. Wu et al. (1992) Nucl. Med. Biol.,19(2): 239-244 discloses a synthesis of macrocylic chelating agents forradiolabeling proteins with ¹¹¹IN and ⁹⁰Y. Wu et al. makes a labeledDOTA-biotin conjugate to study the stability and biodistribution ofconjugates with avidin, a model protein for studies. This conjugate wasmade using a biotin hydrazide which contained a free amino group toreact with an in situ generated activated DOTA derivative.

Cytotoxins.

The macropinocytosis pathway internalizing antibodies described hereincan be used to deliver a variety of cytotoxic drugs includingtherapeutic drugs, a compound emitting radiation, molecules of plants,fungal, or bacterial origin, biological proteins, and mixtures thereof.The cytotoxic drugs can be intracellularly acting cytotoxic drugs, suchas short-range radiation emitters, including, for example, short-range,high-energy α-emitters as described above.

Enzymatically active toxins and fragments. thereof are exemplified bydiphtheria toxin A fragment, nonbinding active fragments of diphtheriatoxin, exotoxin A (from Pseudomonas aeruginosa), ricin A chain, abrin Achain, modeccin A chain, .alpha.-sacrin, certain Aleurites fordiiproteins, certain Dianthin proteins, Phytolacca americana proteins (PAP,PAPII and PAP-S), Morodica charantia inhibitor, curcin, crotin,Saponaria officinalis inhibitor, gelonin, mitogillin, restrictocin,phenomycin, enomycin, and the tricothecenes, for example. A variety ofradionuclides are available for the production of radioconjugatedantibodies. Examples include, but are not limited to ²¹²Bi, ¹³¹I, ¹³¹In,⁹⁰I, ¹⁸⁶Re, and the like.

In certain embodiments the cytotoxins can include, but are not limitedto Pseudomonas exotoxins, Diphtheria toxins, ricin, abrin andderivatives thereof. Pseudomonas exotoxin A (PE) is an extremely activemonomeric protein (molecular weight 66 kD), secreted by Pseudomonasaeruginosa, which inhibits protein synthesis in eukaryotic cells throughthe inactivation of elongation factor 2 (EF-2) by catalyzing itsADP-ribosylation (catalyzing the transfer of the ADP ribosyl moiety ofoxidized NAD onto EF-2).

The toxin contains three structural domains that act in concert to causecytotoxicity. Domain Ia (amino acids 1-252) mediates cell binding.Domain II (amino acids 253-364) is responsible for translocation intothe cytosol and domain III (amino acids 400-613) mediates ADPribosylation of elongation factor 2, which inactivates the protein andcauses cell death. The function of domain Ib (amino acids 365-399)remains undefined, although a large part of it, amino acids 365-380, canbe deleted without loss of cytotoxicity. See Siegall et al. (1989) J.Biol. Chem. 264: 14256-14261.

In certain embodiments the antibody is attached to a preferred moleculein which domain Ia (amino acids 1 through 252) is deleted and aminoacids 365 to 380 have been deleted from domain Ib. In certainembodiments all of domain Ib and a portion of domain II (amino acids 350to 394) can be deleted, particularly if the deleted sequences arereplaced with a linking peptide.

In addition, the PE and other cytotoxic proteins can be further modifiedusing site-directed mutagenesis or other techniques known in the art, toalter the molecule for a particular desired application. For example,means to alter the PE molecule in a manner that does not substantiallyaffect the functional advantages provided by the PE molecules describedhere can also be used and such resulting molecules are intended to becovered herein.

Methods of cloning genes encoding PE fused to various ligands are wellknown to those of skill in the art (see, e.g., Siegall et al. (1989)FASEB J., 3: 2647-2652; and Chaudhary et al. (1987) Proc. Natl. Acad.Sci. USA, 84: 4538-4542).

Like PE, diphtheria toxin (DT) kills cells by ADP-ribosylatingelongation factor 2 thereby inhibiting protein synthesis. Diphtheriatoxin, however, is divided into two chains, A and B, linked by adisulfide bridge. In contrast to PE, chain B of DT, which is on thecarboxyl end, is responsible for receptor binding and chain A, which ispresent on the amino end, contains the enzymatic activity (Uchida et al.(1972) Science, 175: 901-903; Uchida et al. (1973) J. Biol. Chem., 248:3838-3844).

In certain embodiments, the antibody-Diphtheria toxin immunoconjugatesof this invention have the native receptor-binding domain removed bytruncation of the Diphtheria toxin B chain. One illustrative modifiedDipththeria toxin is DT388, a DT in which the carboxyl terminal sequencebeginning at residue 389 is removed (see, e.g., Chaudhary et al. (1991)Bioch. Biophys. Res. Comm., 180: 545-551). Like the PE chimericcytotoxins, the DT molecules can be chemically conjugated to theprostate cancer specific antibody, but, in certain preferredembodiments, the antibody will be fused to the Diphtheria toxin byrecombinant means (see, e.g., Williams et al. (1990) J. Biol. Chem. 265:11885-11889).

Another suitable toxin is saporin. Saporin is a plant toxin that acts asa ribosome-inactivating protein that inhibits protein synthesistypically resulting in cellular apoptosis.

Viral Particles.

In certain embodiments, the effector comprises a viral particle (e.g., afilamentous phage, an adeno-associated virus (AAV), a lentivirus, andthe like). The antibody can be conjugated to the viral particle and/orcan be expressed on the surface of the viral particle (e.g. afilamentous phage). The viral particle can additionally include anucleic acid that is to be delivered to the target (e.g., prostatecancer) cell. The use of viral particles to deliver nucleic acids tocells is described in detail in WO 99/55720, U.S. Pat. Nos. 6,670,188,6,642,051, and 6,669,936. Illustrative nucleic acids include, but arenot limited to siRNAs (e.g., an EphA2 siRNA).

Other suitable effector molecules include pharmacological agents (drugs)or encapsulation systems containing various pharmacological agents.Thus, in various embodiments, it is recognized that the macropinocytosispathway internalizing antibody can be attached directly or through alinker to a drug that is to be delivered directly to the tumor or to anencapsulation system (e.g., a lipid, liposome, microparticle,nanoparticle, dendrimer, etc.) containing the drug. The term “drug”includes any substance that, when administered into the body of a livingorganism, alters normal bodily function. Generally a drug is a substanceused in the treatment, cure, prevention, or diagnosis of disease or usedto otherwise enhance physical or mental well-being. In one embodiment,the drug is an anti-neoplastic and/or cytostatic and/or cytotoxic drug(e.g., an anti-cancer drug).

Anti-cancer drugs are well known to those of skill in the art andinclude, but are not limited to, anti-cancer antibodies (e.g.,trastuzumab (HERCEPTIN®), rituximab (RITUXAN®), etc.), antimetabolites,alkylating agents, topoisomerase inhibitors, microtubule targetingagents, kinase inhibitors, protein synthesis inhibitors, somatostatinanalogs, glucocorticoids, aromatose inhibitors, mTOR inhibitors, proteinKinase B (PKB) inhibitors, phosphatidylinositol, 3-Kinase (PI3K)Inhibitors, cyclin dependent kinase inhibitors, anti-TRAIL molecules,MEK inhibitors, and the like. In certain embodiments the anti-cancercompounds include, but are not limited to flourouracil (5-FU),capecitabine/XELODA, 5-Trifluoromethyl-2′-deoxyuridine, methotrexatesodium, raltitrexed/Tomudex, pemetrexed/Alimta®, cytosine Arabinoside(Cytarabine, Ara-C)/Thioguanine, 6-mercaptopurine (Mercaptopurine,6-MP), azathioprine/Azasan, 6-thioguanine (6-TG)/Purinethol (TEVA),pentostatin/Nipent, fludarabine phosphate/Fludara®, cladribine (2-CdA,2-chlorodeoxyadenosine)/Leustatin, floxuridine (5-fluoro-2)/FUDR(Hospira, Inc.), ribonucleotide Reductase Inhibitor (RNR),cyclophosphamide/Cytoxan (BMS), neosar, ifosfamide/Mitoxana, thiotepa,BCNU-1,3-bis(2-chloroethyl)-1-nitosourea,1,-(2-chloroethyl)-3-cyclohexyl-lnitrosourea, methyl CCNU,hexamethylmelamine, busulfan/Myleran, procarbazine HCL/Matulane,dacarbazine (DTIC), chlorambucil/Leukaran®, melphalan/Alkeran, cisplatin(Cisplatinum, CDDP)/Platinol, carboplatin/Paraplatin,oxaliplatin/Eloxitan, bendamustine, carmustine, chloromethine,dacarbazine (DTIC), fotemustine, lomustine, mannosulfan, nedaplatin,nimustine, prednimustine, ranimustine, satraplatin, semustine,streptozocin, temozolomide, treosulfan, triaziquone, triethylenemelamine, thioTEPA, triplatin tetranitrate, trofosfamide, uramustine,doxorubicin HCL/Doxil, daunorubicin citrate/Daunoxome®, mitoxantroneHCL/Novantrone, actinomycin D, etoposide/Vepesid, topotecanHCL/Hycamtin, teniposide (VM-26), irinotecan HCL(CPT-11)/, Camptosar®,camptothecin, Belotecan, rubitecan, vincristine, vinblastine sulfate,vinorelbine tartrate, vindesine sulphate, paclitaxel/Taxol,docetaxel/Taxotere, nanoparticle paclitaxel, abraxane, ixabepilone,larotaxel, ortataxel, tesetaxel, vinflunine, and the like. In certainembodiments the anti-cancer drug(s) comprise one or more drugs selectedfrom the group consisting of carboplatin (e.g., PARAPLATIN®), Cisplatin(e.g., PLATINOL®, PLATINOL-AQ®), Cyclophosphamide (e.g., CYTOXAN®,NEOSAR®), Docetaxel (e.g., TAXOTERE®), Doxorubicin (e.g., ADRIAMYCIN®),Erlotinib (e.g., TARCEVA®), Etoposide (e.g., VEPESID®), Fluorouracil(e.g., 5-FU®), Gemcitabine (e.g., GEMZAR®), imatinib mesylate (e.g.,GLEEVEC®), Irinotecan (e.g., CAMPTOSAR®), Methotrexate (e.g., FOLEX®,MEXATE®, AMETHOPTERIN®), Paclitaxel (e.g., TAXOL®, ABRAXANE®), Sorafinib(e.g., NEXAVAR®), Sunitinib (e.g., SUTENT®), Topotecan (e.g.,HYCAMTIN®), Vinblastine (e.g., VELBAN®), Vincristine (e.g., ONCOVIN®,VINCASAR PFS®). In certain embodiments the anti-cancer drug comprisesone or more drugs selected from the group consisting of retinoic acid, aretinoic acid derivative, doxirubicin, vinblastine, vincristine,cyclophosphamide, ifosfamide, cisplatin, 5-fluorouracil, a camptothecinderivative, interferon, tamoxifen, and taxol. In certain embodiments theanti-cancer compound is selected from the group consisting of abraxane,doxorubicin, pamidronate disodium, anastrozole, exemestane,cyclophosphamide, epirubicin, toremifene, letrozole, trastuzumab,megestroltamoxifen, paclitaxel, docetaxel, capecitabine, goserelinacetate, zoledronic acid, vinblastine, etc.), an antisense molecule, anSiRNA, and the like.

In certain embodiments, the drug is a tubulin inhibitor. In certainembodiments the tubulin inhibitor is selected from the group consistingof an auristatin; and a maytansine derivative. In certain embodimentsthe drug is an auristatin. Auristatins include synthetic derivatives ofthe naturally occurring compound Dolastatin-10. Auristatins are a familyof antineoplastic/cytostatic pseudopeptides. Dolastatins arestructurally unique due to the incorporation of four unusual amino acids(Dolavaine, Dolaisoleuine, Dolaproine, and Dolaphenine) identified inthe natural biosynthetic product. In addition this class of naturalproduct has numerous asymmetric centets defined by total synthesisstudies by Pettit et al (U.S. Pat. No. 4,978,744). It would appear fromstructure activity relationships that the Dolaisoleuine and Dolaproineresidues appear necessary for antineoplastic activity (U.S. Pat. Nos.5,635,483 and 5,780,588).

In one illustrative, but non-limiting embodiment, the auristatin isselected from the group consisting of auristatin E (AE),monomethylauristatin E (MMAE), auristatin F (MMAF), vcMMAE, and vcMMAF.

In certain embodiments the drug is a maytansine or a structural analogueof maytansine. Maytansines include structurally complex antimitoticpolyketides. Maytansines are potent inhibitors of microtubulin assemblywhich promotes apoptosis in tumor cells. In certain embodiments themaytansine is selected from the group consisting of mertansine (DM1),and a structural analogue of maytansine such as DM3 or DM4. In certainembodiments the drug is mertansine (DM1).

In certain embodiments the drug is DNA interacting agent. In certainembodiments the drug is a DNA interacting agent selected from the groupconsisting of: (a) calicheamicins, duocarmycins, andpyrrolobenzodiazepines (PBDs).

In certain embodiments the drug is a calicheamicin. Calicheamicin is apotent cytotoxic agent that causes double-strand DNA breaks, resultingin cell death. Calicheamicin is a naturally occurring enediyneantibiotic (see, e.g., Smith et al. (1996) J. Med. Chem., 39:2103-2117). In certain embodiments the the calicheamicin iscalicheamicin gamma 1.

In certain embodiments the drug is a duocarmycin. Duocarmycins arepotent anti-tumor antibiotics that exert their biological effectsthrough binding sequence-selectively in the minor groove of DNA duplexand alkylating the N3 of adenine (see, e.g., Boger (1994) Pure & Appl.Chem., 66(4): 837-844). In certain embodiments the duocarmycin isselected from the group consisting of duocarmycin A, duocarmycin B1,duocarmycin B2, duocarmycin C1, duocarmycin C2, duocarmycin D,duocarmycin SA, cyclopropylbenzoindole (CBI) duocarmycin, centanamycin,rachelmycin (CC-1065), adozelesin, bizelesin, and Carzelesin.

In certain embodiments the drug is a pyrrolobenzodiazepine.Pyrrolobenzodiazepines (PBDs) are a class of naturally occurringanti-tumor antibiotics. PBDs exert their anti-tumor activity bycovalently binding to the DNA in the minor groove specifically atpurine-guanine-purine units. They insert on to the N2 of guamine via anaminal linkage and, due to their shape, they cause minimal disruption tothe DNA helix. It is believed that the formation of the DNA-PBD adductinhibits nucleic acid synthesis and causes excision-dependent single anddouble stranded breaks in the DNA helix. As synthetic derivatives thejoining of two PBD units together via a flexible polymethylene tetherallows the PBD dimers to cross-link opposing DNA strands producinghighly lethal lesions.

In certain embodiments, the drug is a synthetic derivative of twopyrrolobenzodiazepines units joined together via a flexiblepolymethylene tether. In certain embodiments the pyrrolobenzodiazepineis selected from the group consisting of anthramycin (and dimersthereof), mazethramycin (and dimers thereof), tomaymycin (and dimersthereof), prothracarcin (and dimers thereof), chicamycin (and dimersthereof), neothramycin A (and dimers thereof), neothramycin B (anddimers thereof), DC-81 (and dimers thereof), Sibiromycin (and dimersthereof), porothramycin A (and dimers thereof), porothramycin B (anddimers thereof), sibanomycin (and dimers thereof), abbeymycin (anddimers thereof), SG2000, and SG2285.

In certain embodiments the effector comprises an encapsulation system,such as a viral capsid, a microporous nanoparticle (e.g., a silica orpolymer nanoparticle), a dendrimer, a lipid, a liposome, or micelle thatcontains a therapeutic composition such as a drug (e.g., any one or moreof the drugs described above), a nucleic acid (e.g. an antisense nucleicacid or another nucleic acid to be delivered to the cell), or anothertherapeutic moiety that is preferably shielded from direct exposure tothe circulatory system. Means of preparing lipids, liposomes,dendrimers, and nanoparticles attached to antibodies are well known tothose of skill in the art (see, e.g., U.S. Pat. No. 4,957,735, Connor etal. (1985) Pharm. Ther., 28: 341-365, and the like).

B) Attachment of the Antibody to the Effector.

One of skill will appreciate that the macropinocytosis pathwayinternalizing antibodies described herein and the effector molecule(s)can be joined together in any order. Thus, where antibody is a singlechain polypeptide, the effector molecule can be joined to either theamino or carboxy termini of the targeting molecule. The antibody canalso be joined to an internal region of the effector molecule, orconversely, the effector molecule can be joined to an internal locationof the antibody, as long as the attachment does not interfere with therespective activities of the molecules.

The antibody and the effector can be attached by any of a number ofmeans well known to those of skill in the art. Typically the effector isconjugated, either directly or through a linker (spacer), to theantibody. However, in certain embodiments, where both the effectormolecule is or comprises a polypeptide it is preferable to recombinantlyexpress the chimeric molecule as a single-chain fusion protein.

Conjugation of the Effector Molecule to the Antibody.

In one embodiment, the macropinocytosis pathway internalizing antibodyis chemically conjugated to the effector molecule (e.g., a cytotoxin, alabel, a ligand, or a drug or liposome, etc.). Means of chemicallyconjugating molecules are well known to those of skill.

The procedure for attaching an effector to an antibody will varyaccording to the chemical structure of the effector and/or antibody.Polypeptides typically contain variety of functional groups; e.g.,carboxylic acid (COOH) or free amine (—NH₂) groups, that are availablefor reaction with a suitable functional group on an effector molecule tobind the effector thereto.

Alternatively, the antibody and/or the effector can be derivatized toexpose or attach additional reactive functional groups. Thederivatization can involve attachment of any of a number of linkermolecules such as those available from Pierce Chemical Company, RockfordIll.

A “linker”, as used herein, is a molecule that is used to join thetargeting molecule to the effector molecule. The linker is capable offorming covalent bonds to both the targeting molecule and to theeffector molecule. Suitable linkers are well known to those of skill inthe art and include, but are not limited to, straight or branched-chaincarbon linkers, heterocyclic carbon linkers, or peptide linkers. Wherethe targeting molecule and the effector molecule are polypeptides, thelinkers may be joined to the constituent amino acids through their sidegroups (e.g., through a disulfide linkage to cysteine). However, in apreferred embodiment, the linkers will be joined to the alpha carbonamino or carboxyl groups of the terminal amino acids.

The immunoconjugates can be made using a variety of bifunctional proteincoupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate(SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters(such as dimethyl adipimidate HCL), active esters (such asdisuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azidocompounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazoniumderivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),diisocyanates (such as tolyene 2,6-diisocyanate), and bis-activefluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). Forexample, a ricin immunotoxin can be prepared as described in Vitetta etal., Science 238: 1098 (1987). Carbon-14-labeled1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid(MX-DTPA) is an exemplary chelating agent for conjugation ofradionucleotide to the antibody (see, e.g., WO94/11026).

Many procedures and linker molecules for attachment of various compoundsincluding radionuclide metal chelates, toxins and drugs to proteins suchas antibodies are known (see, e.g., European Patent Application No.188,256; U.S. Pat. Nos. 4,671,958, 4,659,839, 4,414,148, 4,699,784;4,680,338; 4,569,789; and 4,589,071; and Borlinghaus et al. (1987)Cancer Res. 47: 4071-4075). In particular, production of variousimmunotoxins is well-known within the art and can be found, for examplein Thorpe et al. (1982) Monoclonal Antibodies in Clinical Medicine,Academic Press, pp. 168-190, Waldmann (1991) Science, 252: 1657, U.S.Pat. Nos. 4,545,985 and 4,894,443, and the like.

In some circumstances, it is desirable to free the effector from theantibody when the immunoconjugate has reached its target site.Therefore, immunoconjugates comprising linkages that are cleavable inthe vicinity of the target site may be used when the effector is to bereleased at the target site. Cleaving of the linkage to release theagent from the antibody may be prompted by enzymatic activity orconditions to which the immunoconjugate is subjected either inside thetarget cell or in the vicinity of the target site. When the target siteis a tumor, a linker which is cleavable under conditions present at thetumor site (e.g. when exposed to tumor-associated enzymes or acidic pH)may be used.

A number of different cleavable linkers are known to those of skill inthe art. See U.S. Pat. Nos. 4,618,492; 4,542,225, and 4,625,014. Themechanisms for release of an agent from these linker groups include, forexample, irradiation of a photolabile bond and acid-catalyzedhydrolysis. U.S. Pat. No. 4,671,958, for example, includes a descriptionof immunoconjugates comprising linkers which are cleaved at the targetsite in vivo by the proteolytic enzymes of the patient's complementsystem. In view of the large number of methods that have been reportedfor attaching a variety of radiodiagnostic compounds, radiotherapeuticcompounds, drugs, toxins, and other agents to antibodies one skilled inthe art will be able to determine a suitable method for attaching agiven agent to an antibody or other polypeptide.

Collimation of Chelates.

In certain embodiments, the effector comprises a chelate that isattached to an antibody or to an epitope tag. The the macropinocytosispathway internalizing antibody bears a corresponding epitope tag orantibody so that simple contacting of the antibody to the chelateresults in attachment of the antibody with the effector. The combiningstep can be performed before the moiety is used (targeting strategy) orthe target tissue can be bound to the antibody before the chelate isdelivered. Methods of producing chelates suitable for coupling tovarious targeting moieties are well known to those of skill in the art(see, e.g., U.S. Pat. Nos. 6,190,923, 6,187,285, 6,183,721, 6,177,562,6,159,445, 6,153,775, 6,149,890, 6,143,276, 6,143,274, 6,139,819,6,132,764, 6,123,923, 6,123,921, 6,120,768, 6,120,751, 6,117,412,6,106,866, 6,096,290, 6,093,382, 6,090,800, 6,090,408, 6,088,613,6,077,499, 6,075,010, 6,071,494, 6,071,490, 6,060,040, 6,056,939,6,051,207, 6,048,979, 6,045,821, 6,045,775, 6,030,840, 6,028,066,6,022,966, 6,022,523, 6,022,522, 6,017,522, 6,015,897, 6,010,682,6,010,681, 6,004,533, and 6,001,329).

Production of Fusion Proteins.

Where the antibody and/or the effector is relatively short (i.e., lessthan about 50 amino acids) they can be synthesized using standardchemical peptide synthesis techniques. Where both molecules arerelatively short the chimeric molecule may be synthesized as a singlecontiguous polypeptide. Alternatively the targeting molecule and theeffector molecule may be synthesized separately and then fused bycondensation of the amino terminus of one molecule with the carboxylterminus of the other molecule thereby forming a peptide bond.Alternatively, the targeting and effector molecules can each becondensed with one end of a peptide spacer molecule thereby forming acontiguous fusion protein.

Solid phase synthesis in which the C-terminal amino acid of the sequenceis attached to an insoluble support followed by sequential addition ofthe remaining amino acids in the sequence is the preferred method forthe chemical synthesis of the polypeptides of this invention. Techniquesfor solid phase synthesis are described by Barany and Merrifield,Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides: Analysis,Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, PartA., Merrifield, et al. J. Am. Chem. Soc., 85: 2149-2156 (1963), andStewart et al., Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co.,Rockford, Ill. (1984).

In certain embodiments, the chimeric fusion proteins of the presentinvention are synthesized using recombinant DNA methodology. Generallythis involves creating a DNA sequence that encodes the fusion protein,placing the DNA in an expression cassette under the control of aparticular promoter, expressing the protein in a host, isolating theexpressed protein and, if required, renaturing the protein.

DNA encoding the fusion proteins of this invention can be prepared byany suitable method, including, for example, cloning and restriction ofappropriate sequences or direct chemical synthesis by methods such asthe phosphotriester method of Narang et al. (1979) Meth. Enzymol. 68:90-99; the phosphodiester method of Brown et al. (1979) Meth. Enzymol.68: 109-151; the diethylphosphoramidite method of Beaucage et al. (1981)Tetra. Lett., 22: 1859-1862; and the solid support method of U.S. Pat.No. 4,458,066.

Chemical synthesis produces a single stranded oligonucleotide. This canbe converted into double stranded DNA by hybridization with acomplementary sequence, or by polymerization with a DNA polymerase usingthe single strand as a template. One of skill would recognize that whilechemical synthesis of DNA is limited to sequences of about 100 bases,longer sequences can be obtained by the ligation of shorter sequences.

Alternatively, subsequences can be cloned and the appropriatesubsequences cleaved using appropriate restriction enzymes. Thefragments can then be ligated to produce the desired DNA sequence.

In certain embodiments DNA encoding fusion proteins of the presentinvention can be cloned using PCR cloning methods.

While the antibody and the effector are, in certain embodiments,essentially joined directly together, one of skill will appreciate thatthe molecules can be separated by a spacer, e.g., a peptide spacerconsisting of one or more amino acids (e.g., (Gly₄Ser)₃, SEQ ID NO:10).Generally the spacer will have no specific biological activity otherthan to join the proteins or to preserve some minimum distance or otherspatial relationship between them. However, the constituent amino acidsof the spacer may be selected to influence some property of the moleculesuch as the folding, net charge, or hydrophobicity.

The nucleic acid sequences encoding the fusion proteins can be expressedin a variety of host cells, including E. coli, other bacterial hosts,yeast, and various higher eukaryotic cells such as the COS, CHO and HeLacells lines and myeloma cell lines. The recombinant protein gene will beoperably linked to appropriate expression control sequences for eachhost.

The plasmids of the invention can be transferred into the chosen hostcell by well-known methods such as calcium chloride transformation forE. coli and calcium phosphate treatment or electroporation for mammaliancells. Cells transformed by the plasmids can be selected by resistanceto antibiotics conferred by genes contained on the plasmids, such as theamp, gpt, neo and hyg genes.

Once expressed, the recombinant fusion proteins can be purifiedaccording to standard procedures of the art, including ammonium sulfateprecipitation, affinity columns, column chromatography, gelelectrophoresis and the like (see, generally, R. Scopes (1982) ProteinPurification, Springer-Verlag, N.Y.; Deutscher (1990) Methods inEnzymology Vol. 182: Guide to Protein Purification., Academic Press,Inc. N.Y.). Substantially pure compositions of at least about 90 to 95%homogeneity are preferred, and 98 to 99% or more homogeneity are mostpreferred for pharmaceutical uses. Once purified, partially or tohomogeneity as desired, the polypeptides may then be usedtherapeutically.

One of skill in the art would recognize that after chemical synthesis,biological expression, or purification, the fusion protein may possess aconformation substantially different than the native conformations ofthe constituent polypeptides. In this case, it may be necessary todenature and reduce the polypeptide and then to cause the polypeptide tore-fold into the preferred conformation. Methods of reducing anddenaturing proteins and inducing re-folding are well known to those ofskill in the art (see, e.g., Debinski et al. (1993) J. Biol. Chem., 268:14065-14070; Kreitman and Pastan (1993) Bioconjug. Chem., 4: 581-585;and Buchner, et al. (1992) Anal. Biochem., 205: 263-270).

One of skill would recognize that modifications can be made to thefusion proteins without diminishing their biological activity. Somemodifications may be made to facilitate the cloning, expression, orincorporation of the targeting molecule into a fusion protein. Suchmodifications are well known to those of skill in the art and include,for example, a methionine added at the amino terminus to provide aninitiation site, or additional amino acids placed on either terminus tocreate conveniently located restriction sites or termination codons.

Pharmaceutical Compositions.

The macropinocytosis pathway internalizing antibodies described hereinand/or immunoconjugates thereof are useful for parenteral, topical,oral, or local administration (e.g. injected into a tumor site), aerosoladministration, or transdermal administration, for prophylactic, butprincipally for therapeutic treatment. The pharmaceutical compositionscan be administered in a variety of unit dosage forms depending upon themethod of administration. For example, unit dosage forms suitable fororal administration include powder, tablets, pills, capsules andlozenges. It is recognized that the antibodies described herein and/orimmunoconjugates thereof and pharmaceutical compositions comprisingantibodies described herein and/or immunoconjugates thereof, whenadministered orally, are preferably protected from digestion. This canbe accomplished by a number of means known to those of skill in the art,e.g., by complexing the protein with a composition to render itresistant to acidic and enzymatic hydrolysis or by packaging the proteinin an appropriately resistant carrier such as a liposome. Means ofprotecting proteins from digestion are well known in the art.

In various embodiments a composition, e.g., a pharmaceuticalcomposition, containing one or a combination of the macropinocytosispathway internalizing antibodies, or antigen-binding portion(s) thereof,or immunoconjugates thereof, formulated together with a pharmaceuticallyacceptable carrier are provided.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onthe route of administration, the active compound, i.e., antibody,immunoconjugate, may be coated in a material to protect the compoundfrom the action of acids and other natural conditions that mayinactivate the compound.

In certain embodiments the antibody and/or immunoconjugate can beadministered in the “native” form or, if desired, in the form of salts,esters, amides, prodrugs, derivatives, and the like, provided the salt,ester, amide, prodrug or derivative is suitable pharmacologically, i.e.,effective in the present method(s). Salts, esters, amides, prodrugs andother derivatives of the active agents can be prepared using standardprocedures known to those skilled in the art of synthetic organicchemistry and described, for example, by March (1992) Advanced OrganicChemistry; Reactions, Mechanisms and Structure, 4th Ed. N.Y.Wiley-Interscience, and as described above.

By way of illustration, a pharmaceutically acceptable salt can beprepared for any of the antibodies and/or immunoconjugates describedherein having a functionality capable of forming a salt. Apharmaceutically acceptable salt is any salt that retains the activityof the parent compound and does not impart any deleterious or untowardeffect on the subject to which it is administered and in the context inwhich it is administered.

In various embodiments pharmaceutically acceptable salts may be derivedfrom organic or inorganic bases. The salt may be a mono or polyvalention. Of particular interest are the inorganic ions, lithium, sodium,potassium, calcium, and magnesium. Organic salts may be made withamines, particularly ammonium salts such as mono-, di- and trialkylamines or ethanol amines. Salts may also be formed with caffeine,tromethamine and similar molecules.

Methods of formulating pharmaceutically active agents as salts, esters,amide, prodrugs, and the like are well known to those of skill in theart. For example, salts can be prepared from the free base usingconventional methodology that typically involves reaction with asuitable acid. Generally, the base form of the drug is dissolved in apolar organic solvent such as methanol or ethanol and the acid is addedthereto. The resulting salt either precipitates or can be brought out ofsolution by addition of a less polar solvent. Suitable acids forpreparing acid addition salts include, but are not limited to bothorganic acids, e.g., acetic acid, propionic acid, glycolic acid, pyruvicacid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid,fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid,mandelic acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid, and the like, as well asinorganic acids, e.g., hydrochloric acid, hydrobromic acid, sulfuricacid, nitric acid, phosphoric acid, and the like. An acid addition saltcan be reconverted to the free base by treatment with a suitable base.Certain particularly preferred acid addition salts of the active agentsherein include halide salts, such as may be prepared using hydrochloricor hydrobromic acids. Conversely, preparation of basic salts of theactive agents of this invention are prepared in a similar manner using apharmaceutically acceptable base such as sodium hydroxide, potassiumhydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine, or thelike. Particularly preferred basic salts include alkali metal salts,e.g., the sodium salt, and copper salts.

For the preparation of salt forms of basic drugs, the pKa of thecounterion is preferably at least about 2 pH units lower than the pKa ofthe drug. Similarly, for the preparation of salt forms of acidic drugs,the pKa of the counterion is preferably at least about 2 pH units higherthan the pKa of the drug. This permits the counterion to bring thesolution's pH to a level lower than the pH_(max) to reach the saltplateau, at which the solubility of salt prevails over the solubility offree acid or base. The generalized rule of difference in pKa units ofthe ionizable group in the active pharmaceutical ingredient (API) and inthe acid or base is meant to make the proton transfer energeticallyfavorable. When the pKa of the API and counterion are not significantlydifferent, a solid complex may form but may rapidly disproportionate(i.e., break down into the individual entities of drug and counterion)in an aqueous environment.

Preferably, the counterion is a pharmaceutically acceptable counterion.Suitable anionic salt forms include, but are not limited to acetate,benzoate, benzylate, bitartrate, bromide, carbonate, chloride, citrate,edetate, edisylate, estolate, fumarate, gluceptate, gluconate,hydrobromide, hydrochloride, iodide, lactate, lactobionate, malate,maleate, mandelate, mesylate, methyl bromide, methyl sulfate, mucate,napsylate, nitrate, pamoate (embonate), phosphate and diphosphate,salicylate and disalicylate, stearate, succinate, sulfate, tartrate,tosylate, triethiodide, valerate, and the like, while suitable cationicsalt forms include, but are not limited to aluminum, benzathine,calcium, ethylene diamine, lysine, magnesium, meglumine, potassium,procaine, sodium, tromethamine, zinc, and the like.

Preparation of esters typically involves functionalization of hydroxyland/or carboxyl groups that are present within the molecular structureof the antibody and/or immunoconjugate. In certain embodiments, theesters are typically acyl-substituted derivatives of free alcoholgroups, i.e., moieties that are derived from carboxylic acids of theformula RCOOH where R is alky, and preferably is lower alkyl. Esters canbe reconverted to the free acids, if desired, by using conventionalhydrogenolysis or hydrolysis procedures.

Amides can also be prepared using techniques known to those skilled inthe art or described in the pertinent literature. For example, amidesmay be prepared from esters, using suitable amine reactants, or they maybe prepared from an anhydride or an acid chloride by reaction withammonia or a lower alkyl amine.

Pharmaceutical compositions comprising the antibodies and/orimmunoconjugates described herein can be administered alone or incombination therapy, i.e., combined with other agents. For example, thecombination therapy can include a an antibody or immunoconjugate with atleast one or more additional therapeutic agents, such as the anti-canceragents described infra. The pharmaceutical compositions can also beadministered in conjunction with radiation therapy and/or surgery.

A composition comprising the antibodies and/or immunoconjugatesdescribed herein can be administered by a variety of methods known inthe art. As will be appreciated by the skilled artisan, the route and/ormode of administration will vary depending upon the desired results. Theactive compounds can be prepared with carriers that will protect thecompound against rapid release, such as a controlled releaseformulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art (see, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978).

In certain embodiments administration of the macropinocytosis pathwayinternalizing antibody or immunoconjugate may be facilitated by coatingthe antibody or immunoconjugate composition, or co-administering theantibody or immunoconjugate, a material to prevent its inactivation. Forexample, the compound may be administered to a subject in an appropriatecarrier, for example, liposomes, or a diluent. Pharmaceuticallyacceptable diluents include, but are not limited to, saline and aqueousbuffer solutions. Liposomes include, but are not limited to,water-in-oil-in-water CGF emulsions as well as conventional liposomes(Strejan et al. (1984) J. Neuroimmunol, 7: 27).

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofis contemplated. Supplementary active compounds can also be incorporatedinto the compositions.

In various embodiments the therapeutic compositions are typicallysterile and stable under the conditions of manufacture and storage. Thecomposition(s) can be formulated as a solution, a microemulsion, in alipid or liposome, or other ordered structure suitable to contain highdrug concentration(s). In certain embodiments the carrier can be asolvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), and suitable mixtures thereof. The properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. In many cases, it will bepreferable to include isotonic agents, for example, sugars, polyalcoholssuch as mannitol, sorbitol, or sodium chloride in the composition.Prolonged absorption of the injectable compositions can be brought aboutby including in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., antibodies and/or immunoconjugates described herein) inthe required amount in an appropriate solvent with one or a combinationof ingredients enumerated above, as required, followed by sterilizationmicrofiltration. Generally, dispersions are prepared by incorporatingthe active compound into a sterile vehicle that contains a basicdispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, illustrative methods of preparationinclude vacuum drying, and freeze-drying (lyophilization) that yield apowder of the active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. For example, in certainembodiments, the antibodies and/or immunoconjugates described herein maybe administered once or twice daily, or once or twice weekly, or once ortwice monthly by subcutaneous injection.

It is especially advantageous to formulate parenteral compositions inunit dosage form for ease of administration and uniformity of dosage.Unit dosage form as used herein refers to physically discrete unitssuited as unitary dosages for the subjects to be treated. Each unitcontains a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specifications for the unit dosage forms aredictated by and directly dependent on (a) the unique characteristics ofthe active compound and the particular therapeutic effect to beachieved, and (b) the limitations inherent in the art of compoundingsuch an active compound for the treatment of individuals.

In certain embodiments the formulation comprises a pharmaceuticallyanti-oxidant. Examples of pharmaceutically-acceptable antioxidantsinclude: (1) water soluble antioxidants, such as ascorbic acid, cysteinehydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfiteand the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate,butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT),lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metalchelating agents, such as citric acid, ethylenediamine tetraacetic acid(EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

For the therapeutic compositions, formulations of the antibodies and/orimmunoconjugates described herein include those suitable for oral,nasal, topical (including buccal and sublingual), rectal, vaginal and/orparenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods knownin the art of pharmacy. The amount of active ingredient which can becombined with a carrier material to produce a single dosage form willvary depending upon the subject being treated, and the particular modeof administration. The amount of active ingredient that can be combinedwith a carrier material to produce a single dosage form will generallybe that amount of the composition which produces a therapeutic effect.Generally, out of one hundred percent, this amount will range from about0.001 percent to about ninety percent of active ingredient, preferablyfrom about 0.005 percent to about 70 percent, most preferably from about0.01 percent to about 30 percent.

Formulations of antibodies and/or immunoconjugates described herein thatare suitable for vaginal administration also include pessaries, tampons,creams, gels, pastes, foams or spray formulations containing suchcarriers as are known in the art to be appropriate. Dosage forms for thetopical or transdermal administration of antibodies and/orimmunoconjugates described herein include powders, sprays, ointments,pastes, creams, lotions, gels, solutions, patches and inhalants. Incertain embodiments the active compound may be mixed under sterileconditions with a pharmaceutically acceptable carrier, and with anypreservatives, buffers, or propellants that may be required.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and include, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection, andinfusion.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions comprising antibodies and/orimmunoconjugates described herein include, but are not limited to water,ethanol, polyols (such as glycerol, propylene glycol, polyethyleneglycol, and the like), and suitable mixtures thereof, vegetable oils,such as olive oil, and injectable organic esters, such as ethyl oleate,and the like. Proper fluidity can be maintained, for example, by the useof coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

In various embodiments these compositions may also contain adjuvantssuch as preservatives, wetting agents, emulsifying agents and dispersingagents. Particular examples of adjuvants that are well-known in the artinclude, for example, inorganic adjuvants (such as aluminum salts, e.g.,aluminum phosphate and aluminum hydroxide), organic adjuvants (e.g.,squalene), oil-based adjuvants, virosomes (e.g., virosomes that containa membrane-bound hemagglutinin and neuraminidase derived from theinfluenza virus).

Prevention of presence of microorganisms in formulations may be ensuredboth by sterilization procedures, and/or by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents that delay absorption such as aluminum monostearate andgelatin.

When the antibodies and/or immunoconjugates described herein areadministered as pharmaceuticals, to humans and animals, they can begiven alone or as a pharmaceutical composition containing, for example,0.001 to 90% (more preferably, 0.005 to 70%, such as 0.01 to 30%) ofactive ingredient in combination with a pharmaceutically acceptablecarrier.

Regardless of the route of administration selected, the antibodiesand/or immunoconjugates described herein, that may be used in a suitablehydrated form, and/or the pharmaceutical compositions, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients (e.g., antibodies and/orimmunoconjugates described herein) in the pharmaceutical compositions ofthe present invention may be varied so as to obtain an amount of theactive ingredient which is effective to achieve the desired therapeuticresponse for a particular patient, composition, and mode ofadministration, without being toxic to the patient. The selected dosagelevel will depend upon a variety of pharmacokinetic factors includingthe activity of the particular compositions of the present inventionemployed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts. A physician orveterinarian having ordinary skill in the art can readily determine andprescribe the effective amount of the pharmaceutical compositionrequired. For example, the physician or veterinarian could start dosesof the compounds of the invention employed in the pharmaceuticalcomposition at levels lower than that required in order to achieve thedesired therapeutic effect and gradually increase the dosage until thedesired effect is achieved. In general, a suitable daily dose ofantibodies and/or immunoconjugates described herein will be that amountof the compound which is the lowest dose effective to produce atherapeutic effect. Such an effective dose will generally depend uponthe factors described above. In certain embodiments, it is preferredthat administration be intravenous, intramuscular, intraperitoneal, orsubcutaneous, preferably administered proximal to the site of thetarget. If desired, the effective daily dose of a therapeuticcomposition may be administered a single dosage, or as two, three, four,five, six or more sub-doses administered separately at appropriateintervals throughout the day, optionally, in unit dosage forms. While itis possible for antibodies and/or immunoconjugates described herein tobe administered alone, it is typically preferable to administer thecompound(s) as a pharmaceutical formulation (composition).

In certain embodiments the therapeutic compositions can be administeredwith medical devices known in the art. For example, in a illustrativeembodiment, antibodies and/or immunoconjugates described herein can beadministered with a needleless hypodermic injection device, such as thedevices disclosed in U.S. Pat. Nos. 5,399,163, 5,383,851, 5,312,335,5,064,413, 4,941,880, 4,790,824, or 4,596,556. Examples of usefulwell-known implants and modules are described for example in U.S. Pat.No. 4,487,603, which discloses an implantable micro-infusion pump fordispensing medication at a controlled rate, in U.S. Pat. No. 4,486,194,which discloses a therapeutic device for administering medicationsthrough the skin, in U.S. Pat. No. 4,447,233, which discloses amedication infusion pump for delivering medication at a precise infusionrate, in U.S. Pat. No. 4,447,224, which discloses a variable flowimplantable infusion apparatus for continuous drug delivery, in U.S.Pat. No. 4,439,196, which discloses an osmotic drug delivery systemhaving multi-chamber compartments, and in U.S. Pat. No. 4,475,196, whichdiscloses an osmotic drug delivery system. Many other such implants,delivery systems, and modules are known to those skilled in the art.

In certain embodiments, the macropinocytosis pathway internalizingantibodies and/or immunoconjugates described herein can be formulated toensure proper distribution in vivo. For example, the blood-brain barrier(BBB) excludes many highly hydrophilic compounds. To ensure that thetherapeutic compounds of the invention cross the BBB (if desired), theycan be formulated, for example, in liposomes. For methods ofmanufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548;and 5,399,331. The liposomes may comprise one or more moieties which areselectively transported into specific cells or organs, thus enhancetargeted drug delivery (see, e.g., Ranade (1989) J. Clin. Pharmacol. 29:685). Illustrative targeting moieties include, but are not limited tofolate or biotin (see, e.g., U.S. Pat. No. 5,416,016); mannosides(Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153: 1038);antibodies (Bloeman et al. (1995) FEBS Lett. 357:140; Owais et al.(1995) Antimicrob. Agents Chemother. 39:180); surfactant protein Areceptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134).

Kits.

Where a radioactive, or other, effector is used as a diagnostic and/ortherapeutic agent, it is frequently impossible to put the ready-for-usecomposition at the disposal of the user, because of the often poor shelflife of the radiolabeled compound and/or the short half-life of theradionuclide used. In such cases the user can carry out the labelingreaction with the radionuclide in the clinical hospital, physician'soffice, or laboratory. For this purpose, or other purposes, the variousreaction ingredients can then be offered to the user in the form of aso-called “kit”. The kit is preferably designed so that themanipulations necessary to perform the desired reaction should be assimple as possible to enable the user to prepare from the kit thedesired composition by using the facilities that are at his disposal.Therefore the invention also relates to a kit for preparing acomposition according to this invention.

In certain embodiments, such a kit comprises one or more antibodies orimmumoconjugates described herein. The antibodies or immumoconjugatescan be provided, if desired, with inert pharmaceutically acceptablecarrier and/or formulating agents and/or adjuvants is/are added. Inaddition, the kit optionally includes a solution of a salt or chelate ofa suitable radionuclide (or other active agent), and (iii) instructionsfor use with a prescription for administering and/or reacting theingredients present in the kit.

The kit to be supplied to the user may also comprise the ingredient(s)defined above, together with instructions for use, whereas the solutionof a salt or chelate of the radionuclide, defined sub (ii) above, whichsolution has a limited shelf life, may be put to the disposal of theuser separately.

The kit can optionally, additionally comprise a reducing agent and/or,if desired, a chelator, and/or instructions for use of the compositionand/or a prescription for reacting the ingredients of the kit to formthe desired product(s). If desired, the ingredients of the kit may becombined, provided they are compatible.

In certain embodiments, the immunoconjugate can simply be produced bycombining the components in a neutral medium and causing them to react.For that purpose the effector may be presented to the antibody, forexample, in the form of a chelate.

When kit constituent(s) are used as component(s) for pharmaceuticaladministration (e.g. as an injection liquid) they are preferablysterile. When the constituent(s) are provided in a dry state, the usershould preferably use a sterile physiological saline solution as asolvent. If desired, the constituent(s) may be stabilized in theconventional manner with suitable stabilizers, for example, ascorbicacid, gentisic acid or salts of these acids, or they may comprise otherauxiliary agents, for example, fillers, such as glucose, lactose,mannitol, and the like.

While the instructional materials, when present, typically comprisewritten or printed materials they are not limited to such. Any mediumcapable of storing such instructions and communicating them to an enduser is contemplated by this invention. Such media include, but are notlimited to electronic storage media (e.g., magnetic discs, tapes,cartridges, chips), optical media (e.g., CD ROM), and the like. Suchmedia may include addresses to internet sites that provide suchinstructional materials.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 High-Content Analysis of Antibody Phage-Display LibrariesIdentifies Tumor Selective Macropinocytosis-Dependent RapidlyInternalizing Antibodies

Abbreviations

The following abbreviations are used in this example: HCA: High contentanalysis; ScFv: single chain variable fragment; PCC: Pearson'scorrelation coefficient; CFU: Colony forming unit; MFI: meanfluorescence intensity; EEA: early endosomal antigen; LAMP:lysosomal-associated membrane protein; IgG: immunoglobulin G; ND70-TR:Texas Red-conjugated neutral dextran 70 kDa; FBS: Fetal bovine serum;HEK: human embryonic kidney; LCM: Laser capture microdissection; EphA2:ephrin type-A receptor 2; HRP: horseradish peroxidase; IC50: halfmaximal inhibitory concentration; MAbs: monoclonal antibodies.

Summary of Example 1

Many forms of antibody-based targeted therapeutics, including antibodydrug conjugates, utilize the internalizing function of the targetingantibody to gain intracellular entry into tumor cells. Ideal antibodiesfor developing such therapeutics should be capable of bothtumor-selective binding and efficient endocytosis. The macropinocytosispathway is capable of both rapid and bulk endocytosis, and recentstudies have demonstrated that it is selectively upregulated by cancercells. It was hypothesized that receptor-dependent macropinocytosis canbe achieved using tumor-targeting antibodies that internalize via themacropinocytosis pathway, improving potency and selectivity of theantibody-based targeted therapeutic. While phage antibody displaylibraries have been utilized to find antibodies that bind andinternalize to target cells, it is believed that no methods have beendescribed to screen for antibodies that internalize specifically viamacropinocytosis.

A novel screening strategy to identify phage antibodies that bind andrapidly enter tumor cells via macropinocytosis is described herein. Anautomated microscopic imaging-based, High Content Analysis platform wasused to identify novel internalizing phage antibodies that colocalizewith macropinocytic markers from antibody libraries that we havegenerated previously by laser capture microdissection-based selection,which are enriched for internalizing antibodies binding to tumor cellsin situ residing in their tissue microenvironment (Ruan et al. (2006)Mol. Cell Proteomics. 5: 2364-2373). Full-length human IgG moleculesderived from macropinocytosing phage antibodies retained the ability tointernalize via macropinocytosis, validating our screening strategy. Thetarget antigen for a cross-species binding antibody with a highly activemacropinocytosis activity was identified as ephrin type-A receptor 2.Antibody-toxin conjugates created using this macropinocytosing IgG werecapable of potent and receptor-dependent killing of a panel ofEphA2-positive tumor cell lines in vitro. These studies identify novelmethods to screen for and validate antibodies capable ofreceptor-dependent macropinocytosis, allowing further exploration ofthis highly efficient and tumor-selective internalization pathway fortargeted therapy development.

Materials and Methods

Tissue Culture

Prostate cancer cell lines DU145 and LNCaP, breast cancer cell lineMDA-MB-231, lung cancer cell line A549, cervical cancer cell line HeLa,epidermoid carcinorma cell line A431, and human embryonic kidney (HEK)293A cell line were purchased from the American Type Culture Collection(ATCC). Human foreskin normal fibroblast line Hs27 was purchased fromUCSF Cell Culture Core Facility. Benign prostatic hyperplasia (BPH-1)cells were originally obtained from Dr. Gerald Cunha's lab at UCSF(Hayward et al. (1995) In vitro Cell Dev. Biol. Anim. 31: 14-24) andmaintained in the lab. All cells were grown in high-glucose,L-glutamine, and sodium pyruvate-supplemented complete DMEM (CaissonLabs) with the addition of 10% fetal bovine serum (Fisher Scientific)and penicillin-streptomycin solution (Axenia BioLogix). Cells were grownin 5% CO₂ at 37° C. on tissue culture-treated flasks (BD Biosciences).Cells were passaged utilizing 0.25% trypsin-EDTA (Life Technologies).

Preparation of Phage Antibody Display Library Selection Output forScreening

Phage antibody library selection outputs generated previously byLCM-based selection on prostate tumor tissues (Ruan et al. (2006) Mol.Cell Proteomics, 5: 2364-2373) were streaked onto 2×YT agar platescontaining 12.5 μg/ml tetracycline to yield monoclonal phage antibodies.Individual colonies were inoculated in 2×YT containing 12.5 μg/mltetracycline and grown in deep 96-well plates (Fisher Scientific) at 37°C. with 225 RPM shaking for 18 h. The plates were centrifuged to pelletthe bacteria and supernatants containing phage particles weretransferred into a new 96-well plate for HCA experiments (see below).Positive clones from initial HCA screenings were re-tested usingpurified phage using polyethylene glycol (PEG8000) as previouslydescribed (Ruan et al. (2006) Mol. Cell Proteomics, 5: 2364-2373; Zhu etal. (2010) Mol. Cancer Ther. 9: 2131-2141; An et al. (2008) Mol. CancerTher. 7: 569-578; Liu et al. (2004) Cancer Res. 64: 704-710). Antibodysequences were determined using 96-well plate-based DNA sequencing(Functional Biosciences).

Recombinant Antibody Cloning, Expression, and Purification

For IgG production, heavy and light chain variable fragments weresubcloned into IgG-AbVec (kindly provided by Dr. Patrick Wilson atUniversity of Chicago) γ and λ mammalian expression vectors, aspreviously described, to produce secretable IgG1 antibodies (Smith etal. (2009) Nat. Protoc. 4: 372-384). For scFv-Fc fusion production, scFvwas subcloned from phage into pFUSE-hIgG1 Fc2 (InvivoGen). Mammaliantransfection complexes containing antibody expression DNA andpolyethylenimine (Sigma-Aldrich) in Opti-MEM (Life Technologies) wereadded to HEK 293A cells in the presence of serum-free DMEM containingNutridoma-SP (Roche) and penicillin-streptomycin. Antibody-containingmedia were harvested after 4 days and affinity-purified using protein Aagarose (Pierce/Fisher). Antibody concentrations were determined usingthe BLITZ® Bio-Layer interferometry System (ForteBio).

HCA Screening

Supernatants from 96-well bacterial culture plates (see above) were usedfor initial HCA screening. DU145 cells were seeded in 96-well plates (BDBiosciences) overnight, and incubated with phage-containing supernatantsand 50 μg/ml Texas Red-conjugated 70-kDa neutral dextran (ND70-TR, LifeTechnologies) in DMEM/10% FBS at 37° C. with 5% CO₂ overnight. Cellswere washed 3× with PBS, fixed with 4% paraformaldehyde (Santa CruzBiotechnology) in PBS for 10 min, washed 3× in PBS, and thenpermeabilized in PBS containing 1% fraction V bovine serum albumin(Fisher Scientific) and 0.1% TritonX-100 (Sigma). Phage were detectedwith 3.5 μg/ml biotin-conjugated, rabbit anti-fd bacteriophage(Sigma-Aldrich) for 1 h at RT followed by 1 μg/ml ALEXA FLUOR®488-conjugated streptavidin (Jackson ImmunoResearch) for 15 min at RT.Hoechst 33342 (Thermo Scientific) at 1 μg/ml for 30 min at RT was usedto detect nuclei. The 96-well plates were imaged on a Celllnsight™ NXTHCS platform (Thermo Scientific) with a semi-aprochromat 20× LUCPLFLNobjective (Olympus) utilizing >6 fields per well with a minimum of 300cells per well. Pearson's correlation coefficient analysis betweenND70-TR and phage particles were conducted using Thermo Scientific HCSStudio software suite on all imaged fields and averaged per well.

Confocal Analysis:

DU145 cells were seeded in 8-well Lab-Tek II chambered coverglass(Thermo Scientific) overnight for confocal microscopy studies. Cellswere incubated with antibodies (IgGs at 10 μg/ml or purified phage at10⁹ cfu/ml) and 50 μg/ml ND70-TR in DMEM/10% FBS at 37° C. with 5% CO₂for indicated periods (see text), washed, fixed and permeabilized asdescribed above. To label subcellular structures, rabbit antibodiesagainst early endosomes, lysosomes, caveolin-2, and clathrin heavy chain(Cell Signaling) were added to permeabilized cells at 1:100 dilutionsfor 3 h at RT. Cell-associated human IgGs were detected with 1 μg/mlALEXA FLUOR® 647-conjugated goat anti-human IgG (Jackson ImmunoResearch)for 30 min at RT. Cell-associated phage were detected with 3.5 μg/mlbiotin-conjugated, rabbit anti-fd bacteriophage for 1 h at RT followedby 1 μg/ml ALEXA FLUOR® 488-conjugated streptavidin (JacksonImmunoResearch) for 15 min at RT. Antibodies against organelles weredetected with ALEXA FLUOR® 488- or phycoerythrin-conjugated goatanti-rabbit for 30 min at RT. Hoechst 33342 at 1 μg/ml for 30 min at RTwas used to detect nuclei. Cells in 8-well glass chambered coverglasswere then imaged on the FLUOVIEW® FV10i laser confocal microscope(Olympus) equipped with two galvanometer scanning mirrors. Confocalimages were taken with an Olympus 60X phase contrast water-immersionobjective with NA 1.2. Image analyses including Pearson's and Mander'scorrelation coefficients, Z-projection, Z-projection dissection, and 3Drenderings were performed with the included Olympus confocal softwaresuite.

Internalization Kinetics Assay

DU145 cells seeded in 8-well chambered coverglass were pulsed withantibodies at 10 μg/ml in complete DMEM/FBS for 30 min at 4° C.,followed by a chase in 37° C. warmed, complete DMEM/FBS and incubated at37° C. with 5% CO₂. Individual wells at varying time points were thenwashed in PBS and fixed in 4% paraformaldehyde before undergoingimmunofluorescence as described above. For flow cytometry-basedinternalization kinetics assay, DU145 cells were seeded in 6-well plate,then treated with antibodies at 10 μg/ml for varying amounts of time.Then cells were trypsinized, probed with anti-human secondary antibody,and analyzed on a flow cytometer. Cytochalasin D (Sigma) was resuspendedin DMSO and cells were pulsed with 50 μg/ml of the drug in serum-freeDMEM at 37° C., followed by a chase in complete DMEM/FBS containing thedrug and antibodies.

Immunoprecipitation of the Target Antigen

Purified HCA-F1 scFv-Fc fusions were first chemically cross-linked toprotein A agarose beads. Briefly, antibodies were affinity-bound ontoprotein A agarose (Life Technologies) in a tube. Beads were then spundown and washed with 0.2 M sodium borate, pH 9.0. Dry dimethylpimelimidate (DMP, Sigma) was added to the beads in the presence ofsodium borate to yield a final concentration of 13 mg/ml and incubatedat RT for 30 min. Beads were washed with sodium borate and DMPcrosslinking was repeated a second time. Chemical crosslinking wasterminated through washes with 0.2 M ethanolamine, pH 8.0, for 2 h atRT. Finally, unconjugated antibodies were eluted from beads using 0.1 Mglycine, pH 2.8, followed by washes with PBS. Exposed surface proteinson DU145 cells were biotinylated using EZ-Link Sulfo-NHS-LC-Biotin(Thermo Pierce) according to manufacturer's recommendations and thenlysed using standard RIPA buffer (50 mM Tris, pH 7-8, 150 mM NaCl, 0.1%SDS, 0.5% sodium deoxycholate, 1% NP-40). Immunoprecipitation wasperformed as described previously (Liu et al. (2007) J. Mol. Med.(Berl). 85: 1113-1123; Conrad et al. (2009) J. Mol. Med. (Berl). 87:507-514). Briefly, 5 mg of biotinylated lysates were first pre-clearedagainst protein A agarose for 1 h at RT and then incubated withscFv-Fc-conjugated protein A beads overnight at 4° C. Beads were thenwashed with 500 mM NaCl in PBS, spun down, and boiled in SDS samplebuffer to be run on two 4-12% Tris-glycine SDS-PAGE gels (LifeTechnologies). One gel was GelCode-stained (Thermo) and the other gelwas used for Western blotting using standard procedures. Horseradishperoxidase-conjugated streptavidin was used in the Western Blot toassess which protein band to extract from the GelCode-stained gel.

Antigen Identification by Mass Spectrometry Analysis

Extracted gel bands were trypsin-digested and analyzed via tandem massspectrometry (MS/MS, University of Minnesota) (see, e.g., Table 3).Charge state deconvolution and deisotoping were not performed. All MS/MSsamples were analyzed using Sequest (Thermo Fisher Scientific; version27, rev. 12). Sequest was set up to search thers_human9606_031313_cRAP123 database (unknown version, 36010 entries)assuming the digestion enzyme trypsin. Sequest was searched with afragment ion mass tolerance of 0.80 Da and a parent ion tolerance of0.079 Da and 0.32 Da. Iodoacetamide derivative of cysteine and oxidationof methionine were specified as fixed and variable modifications,respectively, in Sequest. Scaffold (version 4.0.5, Proteome SoftwareInc.) was used to validate protein identifications to create peak lists.Peptide identifications were accepted if they could be established atgreater than 95.0% probability by the Peptide Prophet algorithm (Kelleret al. (2002) Anal. Chem. 74: 5383-5392). Protein identifications wereaccepted if they could be established at greater than 90.0% probabilityand contained at least 2 identified peptides. Protein probabilities wereassigned by the Protein Prophet algorithm (Nesvizhskii et al. (2003)Anal. Chem. 75: 4646-4458). Peptide and protein false discovery rates,as determined by Protein Prophet algorithm, are 0.4% and 0.1%,respectively. Proteins that contained similar peptides and could not bedifferentiated based on MS/MS analysis alone were grouped to satisfy theprinciples of parsimony.

Antibody-Toxin Cytotoxicity Assay

The human IgG HCA-F1 was biotinylated with EZ-Link Sulfo-NHS-LC-Biotin(Thermo Pierce) according to manufacturer's recommendations. A panel oftumor and non-tumorigenic cell lines were seeded in 96-well plates at adensity of 1,000-2,000 cells per well and grown for 16 h at 37° C. in 5%CO₂. Biotinylated IgG HCA-F1 was then incubated with streptavidin-ZAP(saporin conjugated with streptavidin, Advanced Targeting Systems) at amolar ratio of 1:1 and incubated on ice for 30 min to form theantibody-toxin (saporin) conjugate, which was then added to cells andincubated for 96 h at 37° C. in 5% CO₂. Cell viability was thendetermined by CCK-8 assay (Dojindo) according to manufacturer'srecommendations using the Synergy HT microtiter plate reader (Bio-Tek).IC50 values were determined by curve fit using Prism (GraphPadSoftware).

Results

HCA-Based Screening Strategy

The HCA-based strategy that we used to identify antibodies capable ofinternalizing into tumor cells via macropinocytosis is outlined in FIG.2A. The key feature is the development of an HCA platform that allowsquantitative measurement of colocalization between phage antibodies anda macropinocytic marker, ND70-TR. To identify clinically relevantmacropinocytosing antibodies, we screened phage antibody libraries thatwe have generated previously by laser capture microdissection(LCM)-based selection, which are highly enriched for internalizingantibodies that bind to prostate tumor cells in situ residing in thetumor tissue microenvironment (Ruan et al. (2006) Mol. Cell Proteomics.5: 2364-2373).

Analysis of Phage Antibody Binding Patterns by Automated FluorescenceMicroscopy

Phage infected bacteria were arrayed into 96-well plates andphage-containing supernatants were incubated with prostate cancer DU145cells in 96-well plates in the presence of complete DMEM/10% FBS for 24h at 37° C. Phage antibody binding patterns were analyzed by automatedfluorescent microscopy (FIG. 2B). A broad range of patterns ofcell-associated phages was observed but internalization could not beclearly determined (FIG. 10). Image-based quantitation of phage bindingwas performed to generate a mean fluorescence intensity (MFI) value foreach phage antibody (FIG. 2C). We selected the top 25% (MFI>250,000) or360 phage clones for more detailed analysis of internalizing properties(FIG. 2C). FACS analysis of a fraction of these phage clones on DU145cells yielded MFI values consistent with the microscopic imaging-basedanalysis (FIG. 11).

HCA Identifies Phage Antibodies that Internalize Via Macropinocytosis

Previous methods to select and screen for internalizing phage antibodieshave utilized low pH, high salt washes in an attempt to stripsurface-bound phage antibodies. While this approach can be successful,strong binding, high-affinity phage antibodies may be resistant to eventhese harsh conditions. Several of the strongest binding phage antibodyclones were tested on fixed cells, which are incapable ofinternalization, and found that binding was resistant to low pH, highsalt washes (FIG. 12). Thus, new methods are required to identify highaffinity internalizing phage antibodies.

To screen for phage antibody clones that internalize into DU145 cellsvia macropinocytosis, we performed HCA on the strongest binding clones(top 25%, or 360) utilizing ND70-TR as a fluid-phase macropinocyticmarker (Schnatwinkel et al. (2004) PLoS Biol. 2: E261; Veithen et al.(1998) J. Cell Sci. 111(Pt 16): 2329-2335). Previous studies haveestablished that fluorescent high molecular weight dextrans can be usedto label macropinosomes (Schnatwinkel et al. (2004) PLoS Biol. 2: E261).Phage antibody-containing supernatants were co-incubated with ND70-TRover DU145 cells in culture media for 24 h at 37° C. Following washing,fixing and permeabilization, cell-associated phage were detected byanti-phage antibody, and subjected to HCA to assess colocalization withND70-TR (FIG. 3, panel D). An initial image analysis revealed that somephage antibodies internalized into cells and colocalized with ND70-TR,primarily in juxtanuclear structures, while other clones exhibited poorcolocalization with ND70-TR (FIG. 3, panels A, B). Next, a quantitativeanalysis was performed by measuring the Pearson's correlationcoefficient (PCC) between immunolabeled phage and ND70-TR fluorescence.High PCC values identified phage antibodies that exhibited strongcolocalization with ND70-TR, while low PCC values identified phageantibodies that exhibited poor colocalization with ND70-TR (FIG. 3,panel C). About 10%, or 36 clones, possessed greater than 2-fold PCCvalues when compared to controls (FIG. 3, panel E). Followingsequencing, 14 unique antibody sequences were identified from the 36clones.

Endocytosed Phages Macropinocytose En Route to Lysosomes in DU145 Cells

We further characterized three phage antibody clones, named HCA-F1,HCA-M1, and HCA-S1, two of which possessed high (HCA-F1 andHCA-M1, >2-fold PCC values over control) and one with low (HCA-S1,<2-fold PCC value over control) correlation between immunolabeled phagesand ND70-TR. Using fluorescent confocal microscopy, it was determinedwhether these clones could internalize into juxtanuclear structurescoinciding with lysosomal markers. After 24 h incubation with DU145cells, phage antibodies colocalized with lysosomal-associated membraneprotein 1 (LAMP1). Phages HCA-F1 and HCA-M1 were visible as compact,vesicular structures present in a juxtanuclear area while phage HCA-S1exhibited poor internalization (FIG. 4, panel A). Computed 3D tomographyalso demonstrated that endocytosed phage HCA-F1 colocalized withinternalized ND70-TR. It was also determined whether phages could bevisualized within early endosomes during early stages of endocytosis,however, phages did not colocalize with the endosomal marker, earlyendosomal antigen 1 (EEA1) (data not shown), suggesting that either thephages transited quickly through early endosomes or bypassed the earlyendosomes in route to lysosomes.

Phage Macropinocytose into DU145 Cells with Varying Kinetics

It was next determined whether phage antibodies HCA-F1, HCA-M1, andHCA-S1 can exhibit distinguishable internalization kinetics. Whereas twophage antibodies HCA-F1 and HCA-M1 displayed a similar internalizationpattern after a 24 h incubation, only phage HCA-F1 was capable ofinternalizing into DU145 cells after an 8 h incubation (FIG. 4, panelB). PCC analysis between fluorescently immunolabeled, internalizedphages and ND70-TR after an 8 h incubation showed significantdifferences between the three phage antibodies (FIG. 4, panel C).Mander's correlation coefficient analysis, which is similar to PCCanalysis but places weight on fluorescent intensity, also corroboratedthese differences (data not shown).

Internalization of IgGs Derived from scFvs

ScFv from phages HCA-F1, HCA-M1, and HCA-S1 were cloned into full-lengthhuman IgG1 expression constructs and purified IgGs from transientlytransfected human embryonic kidney (HEK) 293A cell supernatants. Thepurified IgGs HCA-F1, HCA-M1, and HCA-S1 demonstrated binding to DU145cells via flow cytometry (FIG. 13) and colocalized with internalizedND70-TR in DU145 cells in a similar fashion to their parental phageantibodies (FIG. 5, panel A). 3D computed tomography showed that IgGHCA-F1 possesses the most robust internalization properties,internalizing almost immediately upon incubation with cells and yieldingvery low amounts of detectable IgG on the surface of the cell after 90minutes of incubation. Similar to the data from the phage experiments,the PCC value between immunolabeled IgG and ND70-TR was significantlyhigher for IgG HCA-F1 than either IgG HCA-M1 or IgG HCA-S1 (FIG. 5,panel B).

Immunolabeling against the endocytic markers EEA1 and LAMP1 was utilizedto examine the colocalization of IgGs HCA-F1, HCA-M1, and HCA-S1 withearly endosomes and lysosomes over varying time intervals. All of theIgGs bound to the surface of DU145 cells almost immediately afteradministration (FIG. 6, panels A-B). IgG HCA-F1 fluorescence increasedin intensity over time in punctate-like structures at the expense ofcell surface fluorescence (FIG. 6, panels A-B). IgG HCA-F1 addition alsoled to increased numbers of EEA1-labeled punctate structures whencompared to either IgGs HCA-M1 or -S1 (FIG. 6, panel A). Antibodycolocalization with both organelles was quantitated via PCC analysisacross all time points. IgG HCA-F1 possessed significantly higher PCCvalues at earlier time points for both EEA1 and LAMP1 when compared toeither IgG HCA-M1 or HCA-S1 (FIG. 6, panels C-D). IgG HCA-F1 did notsignificantly colocalize with caveolin-2 or clathrin heavy chain,especially at earlier time points (FIG. 14). Furthermore, the HCA-F1scFv-Fc fusion also bound, internalized, and colocalized with both EEA1and LAMP1 within DU145 cells in the same fashion as its IgG counterpart(FIG. 15).

IgG HCA-F1 Internalizes Via Macropinocytosis

To confirm antibody internalization via macropinocytosis, antibodyinternalization was studied with and without inhibitors ofmacropinocytosis. Previous studies have demonstrated that cytochalasin Dand ethylisopropylamiloride (EIPA) both inhibit macropinocytosis(Commisso et al. (2013) Nature. 497: 633-637; Gold et al. (2010) PLoSOne. 5: e11360; Veithen et al. (1996) J. Cell Sci. 109(Pt 8): 2005-2012;West et al. (1989) J. Cell Biol. 109: 2731-2739). DU145 cellspre-treated with cytochalasin D, EIPA, or DMSO for 30 min were chasedwith IgG HCA-F1 in the presence of drug or DMSO. Both cytochalasin D andEIPA significantly inhibited IgG HCA-F1 internalization into DU145 cells(FIG. 7, panel A). Measurements of internalized, immunolabeled IgGHCA-F1 fluorescence showed that both cytochalasin D and EIPA decreasedendocytosed IgG HCA-F1 by >50% when compared to DMSO control (FIG. 7,panel B).

EphA2 Identified as Antigen Target for Macropinocytosing IgG HCA-F1

We next sought to determine the target antigen bound by the rapidlyinternalizing macropinocytosing IgG HCA-F1. We surface-biotinylatedDU145 cells, prepared cell lysates and performed immunoprecipitationwith HCA-F1 scFv-Fc immobilized to agarose beads. Immunoprecipitationproducts underwent parallel SDS-PAGE and immunoblotting. Immunoblottingresults with streptavidin-conjugated horseradish peroxidase (HRP) showeda dominant band at ˜110 kDa (FIG. 8, panel A). After excising thecorresponding band from the Coomassie-stained gel, the extracted proteingel slice underwent trypsin-digestion and analysis via tandem massspectrometry. The results identified a transmembrane protein, ephrintype-A receptor 2 (EphA2), as the target antigen (Table 2). For anindependent verification, we ectopically expressed human EphA2 cDNA inChinese hamster ovary (CHO) cells and found that IgG HCA-F1 boundstrongly to these cells but not CHO cells transfected with a controlcDNA (FIG. 8, panels B-C).

TABLE 2 EphA2 identified as the target antigen for IgG HCA-F1. UniqueProtein Gene Size Peptides Sequence Name Name Accession # (kDa) DetectedCoverage % Ephrin EPHA2 NP_004422 108 47 44 type_A receptor 2 precursor

TABLE 3 Mass spectrometry analysis identify peptides of EphA2. SequestDelta Sequest delta Modifi- Ob- Actual Da Delta Strt/ Sequence XCorr Cncations served Mass Chrg (x 10⁻³) PPM TIC Stp  (R)DcNS 2.6496 0.4959Carbamid 693.77 1,385.53 2 -2.477 -1.79 128849 104/ FPGGAS omethyl 116ScK(E) (+57), (SEQ ID Carbamid NO: 11) omethyl (+57) (R)DcNS 2.14810.4668 Carbamid 693.77 1,385.53 2 -3.677 -2.65 20,897.20 104/ FPGGASomethyl 116 ScK(E) (+57), (SEQ ID Carbamid NO: 11) omethyl (+57) (R)DcNS1.9465 0.441 Carbamid 693.77 1,385.53 2 -4.177 -3.01 15,624.10 104/FPGGAS omethyl 116 ScK(E) (+57), (SEQ ID Carbamid NO: 11) omethyl (+57)(R)mHc 4.623 0.529 Oxidation 946.42 2,836.24 3 -2.457 -0.87 67,765.50245/ AVDGE (+16), 268 WLVPI Carbamid GQcLcQ omethyl AGYEK (+57), (V)Carbamid (SEQ ID omethyl NO: 12) (+57), Carbamid omethyl (+57) (R)MHc3.1364 0.575 Carbamid 941.09 2,820.25 3 -4.442 -1.57 31,426.50 245/AVDGE omethyl 268 WLVPI (+57), GQcLcQ Carbamid AGYEK omethyl (V) (+57),(SEQ ID Carbamid NO: 12) omethyl (+57) (K)VED 2.4311 0.422 Carbamid808.35 1,614.68 2  0.5234  0.32 79,332.10 269/ AcQAcS omethyl 282 PGFFK(+57), (F) Carbamid (SEQ ID omethyl NO: 13) (+57) (K)VED 2.3217 0.325Carbamid 808.35 1,614.68 2 -2.077 -1.29 54,619.60 269/ AcQAcS omethyl269 PGFFK (+57), (F) Carbamid (SEQ ID omethyl NO: 14) (+57) (K)FEAS2.3081 0.351 Carbamid 1282.86 3,845.55 3 -9.907 -2.58 33,332.00 283/ESPcLEc omethyl 284 PEHTLP (+57), SPEGAT Carbamid ScEcEE omethyl GFFR(A)(+57), (SEQ ID Carbamid NO: 15) omethyl (+57), Carbamid omethyl (+57)(R)YSEP 2.2298 0.306 386.20 1,155.57 3 -2.346 -2.03 73,404.30 385/ PHGLT394 R(T) (SEQ ID NO: 16) (R)YSEP 2.3917 0.330 578.79 1,155.57 2 -2.146-1.86 113986 385/ PHGLT 394 R(T) (SEQ ID NO: 16) (R)YSEP 2.1526 0.357578.79 1,155.57 2 -1.646 -1.42 45,566.90 385/ PHGLT 394 R(T) (SEQ IDNO: 16) (R)YSEP 2.2759 0.257 386.20 1,155.57 3 -0.746 -0.65 63,530.60385/ PHGLT 394 R(T) (SEQ ID NO: 16) (R)YSEP 2.0678 0.344 578.79 1,155.572  0.354  0.31 47,981.30 385/ PHGLT 394 R(T) (SEQ ID NO: 16) (R)YSEP2.0638 0.280 386.20 1,155.57 3 -2.246 -1.94 494067 385/ PHGLT 394 R(T)(SEQ ID NO: 16) (R)YSEP 2.2515 0.195 386.20 1,155.57 3 -0.546 -0.47122285 385/ PHGLT 394 R(T)* (SEQ ID NO: 16) (R)YSEP 1.9858 0.188 386.201,155.57 3 -2.246 -1.94 207445 385/ PHGLT 394 R(T)** (SEQ ID NO: 16)(R)NGV 2.8737 0.443 495.27 988.5277 2 -2.446 -2.47 314423 416/ SGLVTS425 R(S) (SEQ ID NO: 17) (R)TAS 1.4272 0.314 686.36 1,370.70 2 -3.146-2.29 31,114.50 429/ VSINQT 441 EPPK(V) ** (SEQ ID NO: 18) (R)TAS 3.08430.502 813.94 1,625.86 2 -9.146 -5.62 48,697.80 429/ VSINQT 443 EPPKVR(L) (SEQ ID NO: 19) (R)TAS 2.6076 0.324 542.96 1,625.87 3 -6.946 -4.27263531 429/ VSINQT 443 EPPKVR (L) (SEQ ID NO: 19) (R)STTS 2.7719 0.421979.50 1,956.99 2 -0.246 -0.13 51,078.80 448/ LSVSW 465 SIPPPQ QSR(V)(SEQ ID NO: 20) (R)STTS 2.0494 0.262 979.50 1,956.99 2  1.254  0.6425,873.20 448/ LSVSW 465 SIPPPQ QSR(V)* ** (SEQ ID NO: 20) (R)VWK 2.03190.262 415.22 1,242.64 3 -2.246 -1.81 130884 466/ YEVTY 474 R(K) (SEQ IDNO: 21) (R)KKG 2.8284 0.407 634.32 1,266.63 2  0.254  0.20 27,676.30475/ DSNSY 485 NVR(R) (SEQ ID NO: 22) (R)KKG 1.6464 0.265 423.221,266.63 3 -0.746 -0.59 42,355.60 475/ DSNSY 485 NVR(R) (SEQ ID NO: 22)(R)QSPE 2.3752 0.348 600.28 1,198.55 2  1.454  1.21 189 569/ DVYFS 723578 K(S) (SEQ ID NO: 23) (K)FTTE 3.0577 0.408 Carbamid 724.35 1,446.69 2-0.6113 -0.42 160757 604/ IHPScV omethyl 615 TR(Q) (+57) (SEQ ID NO: 24)(K)FTTE 2.2528 0.213 Carbamid 483.24 1,446.69 3 -0.7113 -0.49 262928604/ IHPScV omethyl 615 TR(Q)** (+57) (SEQ ID NO: 24) (R)QKVI 1.91560.398 762.91 1,523.80 2  2.054  1.35 35,365.50 616/ GAGEF 629 GEVYK (G)(SEQ ID NO: 25) (R)QKVI 1.8738 0.224 508.94 1,523.80 3 -0.346 -0.23200820 616/ GAGEF 629 GEVYK (G)** (SEQ ID NO: 25) (K)VIG 2.5299 0.468634.83 1,267.65 2  2.154  1.70 389857 618/ AGEFG 629 EVYK(G) (SEQ IDNO: 26) (K)QRV 4.3377 0.574 632.08 2,524.28 4  5.354  2.12 65,605.40656/ DFLGE 677 AGIMG QFSHH NIIR(L) (SEQ ID NO: 27) (K)QRV 3.8947 0.552505.86 2,524.28 5  4.154  1.65 77,030.10 656/ DFLGE 677 AGIMG QFSHHNIIR(L) (SEQ ID NO: 27) (K)QRV 3.2965 0.454 Oxidation 509.06 2,540.27 5 1.139  0.45 149402 656/ DFLGE (+16) 677 AGImG QFSHH NIIR(L) (SEQ IDNO: 27) (R)VDF 3.1313 0.412 Oxidation 753.04 2,256.11 3 -0.06101 -0.0354,320.90 658/ LGEAGI (+16) 677 mGQFS HHNIIR (L) (SEQ ID NO: 28) (R)VDF3.2648 0.380 Oxidation 565.03 2,256.11 4 -1.261 -0.56 98,523.40 658/LGEAGI (+16) 677 mGQFS HHNIIR (L) (SEQ ID NO: 28) (R)NILV 2.983 0.142Carbamid 637.35 1,272.69 2  0.9887  0.78 569450 744/ NSNLVc omethyl 754K(V) (+57) (SEQ ID NO: 29) (R)VLE 2.6914 0.508 841.89 1,681.77 2 -3.146-1.87 85,590.20 763/ DDPEA 778 TYTTSG GK(I) (SEQ ID NO: 30) (R)VLE1.8545 0.422 841.89 1,681.77 2 -0.546 -0.32 18,354.50 763/ DDPEA 778TYTTSG GK(I) (SEQ ID NO: 30) (R)VLE 3.3147 0.487 721.37 2,161.09 3-1.246 -0.58 91,933.40 763/ DDPEA 782 TYTTSG GKIPIR (W) (SEQ ID NO: 31)(R)WTA 2.3826 0.300 597.30 1,192.59 2  1.454  1.22 165682 783/ PEAISY792 R(K) (SEQ ID NO: 32) (K)FADI 2.3497 0.408 560.82 1,119.62 2 -1.346-1.20 1045020 864/ VSILDK 873 (L) (SEQ ID NO: 33) (R)VSIR 4.5897 0.550596.99 1,787.95 3 -4.646 -2.60 136449 891/ LPSTSG 907 SEGVPF R(T)(SEQ ID NO: 34) (R)LPST 3.2221 0.440 667.34 1,332.67 2 -0.646 -0.48117106 895/ SGSEG 907 VPFR(T) (SEQ ID NO: 35) (R)LPST 3.0411 0.451667.34 1,332.67 2  0.654  0.49 237650 895/ SGSEG 907 VPFR(T) (SEQ IDNO: 35) (K)VVQ 2.9539 0.429 659.84 1,317.67 2 -3.046 -2.31 36,696.70936/ MTNDD 946 IKR(I) (SEQ ID NO: 36) (K)VVQ 2.3773 0.282 Oxidation667.84 1,333.66 2 -2.161 -1.62 212225 936/ mTNDD (+16) 946 IKR(I)(SEQ ID NO: 36) (R)IAYS 1.9363 0.271 489.30 976.5941 2 -1.746 -1.79145459 958/ LLGLK 966 (D)**** (SEQ ID NO: 37) *Prob 99%; **Prob 95%;***Prob 98%; **** Prob 99%

Receptor-Dependent Macropinocytosis of the Anti-EphA2 IgG

As EphA2 is widely overexpressed by cancer cells (Wykosky and Debinski(2008) Mol. Cancer Res. 6: 1795-1806; Tandon et al. (2011) Expert Opin.Ther. Targets. 15: 31-51), we next examined whether IgG HCA-F1 iscapable of binding to other cancer cell lines and internalizing viamacropinocytosis. We analyzed the binding of IgG HCA-F1 to five humancancer cell lines (prostate cancer DU145, breast cancer MDA-MB-231, lungcancer A549, cervical cancer HeLa, epidermoid carcinoma A431) and twonon-cancer cell lines (Hs27 and BPH-1) by FACS. IgG HCA-F1 binding washigher for all five cancer cell lines when compared to the non-cancercell lines (FIG. 16). IgG HCA-F1 did not bind to the LNCaP line thatdoes not express EphA2 (FIG. 9, panel A), demonstrating thereceptor-dependent nature of this type of cell entry. To assess bindingto cross-species epitopes, we also performed FACS analysis of IgG HCA-F1on a mouse melanoma cell line B16F10 and observed binding, whichsuggests that IgG HCA-F1 bind to an EphA2 epitope conserved acrossspecies (data not shown). To investigate the specificity ofinternalization, IgG HCA-F1 and ND70-TR were co-incubated over theaforementioned panel of both cancer and non-cancer cell lines. Confocalimaging using equal exposure times confirmed that IgG HCA-F1 boundstrongly to cancer cell lines when compared to non-cancer cell lines(FIG. 17, panel A). Internalized IgG HCA-F1 was quantified by measuringmean fluorescent intensities of IgG HCA-F1 within individual, confocalslices of cytosolic areas of cells. Quantitation of internalized IgGHCA-F1 across all cell lines revealed that cancer cell lines possessgreater amounts of internalized IgG HCA-F1 when compared to non-cancercell lines (FIG. 17).

Antibody-Toxin Conjugate Exhibits Potent Cytotoxicity In Vitro

To obtain functional evidence for internalization, we investigatedwhether an IgG HCA-F1-based antibody-toxin conjugate could lead totargeted killing of tumor cells. We created an IgG HCA-F1-toxinconjugate by first modifying IgG HCA-F1 with amine-reactive biotin,followed by attachment of streptavidin-conjugated saporin, a highlypotent ribosome-inactivating protein toxin. Saporin lacks a chainrequired for cell insertion and is thus non-toxic by itself. Theantibody-toxin conjugates were incubated at varying concentrations withboth DU145 (EphA2 positive) and LNCAP (EphA2-negative) cells andexamined cell viability after 4 days. The IgG HCA-F1-toxin conjugateexhibited potent cytotoxicity against DU145 cells (IC₅₀ about 19 pM) butnot on control LNCaP cells (FIG. 9, panel B), demonstrating functionallya receptor-dependent internalization mechanism. Toxin conjugated to acontrol non-binding human IgG did not kill tumor cells, neither didtoxin alone nor naked HCA-F1 IgG. These studies provide functionalevidence for rapid internalization by our anti-EphA2 antibody IgG HCA-F1and demonstrate potential for the development of targeted therapeuticsagainst EphA2-positive tumors.

Discussion

Recent studies suggest that macropinocytosis is a rapid and efficientcellular internalization pathway that is upregulated selectively bytumor cells (Commisso et al. (2013) Nature. 497: 633-637; Reyes-Reyes etal. (2010) Cancer Res. 70: 8617-8629). Exploring this pathway fortargeted therapy development has the potential of improving potency andselectivity for tumor targeting agents. While studies have been donepreviously to identify internalizing antibodies from phage antibodydisplay libraries (Zhu et al. (2010) Mol. Cancer Ther. 9: 2131-2141; Anet al. (2008) Mol. Cancer Ther. 7: 569-578; Liu et al. (2004) CancerRes. 64: 704-710; Poul et al. (2000) J. Mol. Biol. 301: 1149-1161;Rudnick et al. (2011) Cancer Res. 71: 2250-2259), no method has beendeveloped to identify macropinocytosing antibodies. In this study, wedeveloped an HCA-based high throughput method to identifymacropinocytosing antibodies from phage antibody display libraries.Following conversion into full-length human IgGls, we determined byconfocal microscopy that one of the antibodies, IgG HCA-F1, rapidlyinternalizes via macropinocytosis and colocalizes with early endosomeand lysosome markers. The microscopic internalization studies wereconfirmed by functional internalization assays based on the plant toxinsaporin that lacks an internalization mechanism on its own. The rapidinternalization of the HCA-F1 IgG resulted in potent cytotoxicity ofantibody-toxin conjugate against a broad panel of tumor cells expressingthe target antigen, demonstrating functionally that this antibody isefficiently internalized by target cells.

Previous methods to select and screen for internalizing phage antibodieshave utilized low pH, high salt wash buffers in an attempt to strip awaysurface-bound phage antibodies (An et al. (2008) Mol. Cancer Ther. 7:569-578; Liu et al. (2004) Cancer Res. 64: 704-710; Poul et al. (2000)J. Mol. Biol. 301: 1149-1161). While this approach has been at leastpartially successful, strong binding high affinity phage antibodies maybe resistant to even these harsh conditions. Indeed, when we testedstrong binding phage antibodies on fixed cells which are incapable ofinternalization, we found that binding was resistant to low pH, highsalt washes. In addition, we found that analysis of patterns ofcell-associated phage that were generated by non-confocal HCAinstruments was not sufficient to determine if the phage isinternalized. Many heterogeneous patterns were observed, and it wasdifficult to reliably associate any of the patterns withinternalization, let alone macropinocytosis. Thus, our new methods basedon multi-marker microscopic HCA establish an effective means for theidentification of internalizing and furthermore macropinocytosingantibodies from phage display libraries.

Our studies showed that there are major differences in internalizationkinetics between an antibody in soluble form and on phage, which must betaken into consideration for screening design. For example, when testedin full-length IgG or scFv-Fc fusion forms, the highly activemacropinocytosing antibody HCA-F1 starts internalization almostimmediately and completes the process in 40-80 min, while the sameantibody in phage format does so in 8 h. The large size of the phageparticle may have slowed down the internalization process considerablyregardless of how rapidly the antibody internalizes in soluble forms. Inaddition, although in soluble forms different antibodies utilizeddisparate internalization pathways, in phage forms they seem to convergeinto the macropinocytosis pathway. This is not entirely surprisingconsidering the size of the phage particle. Nonetheless, despite thegenerally retarded rate and the near uniform route of phage particleinternalization (phage macropinocytosis), the kinetic differences inphage antibody internalization are a function of the underlying scFv,with HCA-F1-like phage internalizing in 8 h, HCA-M1-like in 24 h andHCA-S1-like >24 h. These kinetics differences allowed us to developscreening schemes to uncover rapidly internalizing antibodies such asthe macropinocytosing antibody HCA-F1. In this context, we would like tore-emphasize that HCA screening using phage directly is convenient andcompatible with the high throughout format, but the result must beverified using antibodies in soluble forms.

Another peculiar feature of phage internalization is revealed by ourorganelle-labeling experiment. While phage antibodies are seen tocolocalize to lysosomes, they could not be readily seen entering cellsvia the early endosomal pathway. We reconciled this observation byhypothesizing that large, macropinocytosed phage particles may betrafficking via endosomes distinct from traditional coatedvesicle-formed endosomes, which has been previously observed (Hewlett etal. (1994) J. Cell Biol. 124: 689-703). For IgG however, we were able toobserve that the phage-derived IgG could internalize viamacropinocytosis towards the lysosomal organelles via the endosomalpathway.

EphA2 is known to be expressed by various tumor cells and play roles intumor invasion and metastasis (Wykosky and Debinski (2008) Mol. CancerRes. 6: 1795-1806). Several groups have developed anti-EphA2 antibodies(Ansuini et al. (2009) J. Oncol. 2009: 951917; Jackson et al. (2008)Cancer Res. 68: 9367-9374; Zhou et al. (2010) J. Mol. Biol. 404: 88-99),and it appears that different epitopes mediate different rates ofinternalization (Ansuini et al. (2009) J. Oncol. 2009: 951917). No phageantibody library selection scheme has been developed previously thatallows for selection of macropinocytosing antibodies binding to EphA2 orother antigens. Our unbiased screening has uncovered an antibody thatbinds to EphA2 and is rapidly internalized by the macropinocytosispathway, thereby creating novel agents against this receptor.

EphA2 has been the target for many forms of cancer therapy development.Nanoparticles conjugated with anti-EphA2 antibodies have been used forsiRNA delivery (Shen et al. (2013) Clin. Cancer Res. 19: 1806-1815;Tanaka et al. (2010) Cancer Res. 70: 3687-3696). In addition, ananti-EphA2 antibody auristatin E conjugate was tested in a phase I trialfor solid tumor treatment (Jackson et al. (2008) Cancer Res. 68:9367-9374; Annunziata et al. (2013) Invest. New Drugs. 31: 77-84). Thisparticular anti-EphA2 antibody-auristatin E-conjugate showedunacceptably high toxicity at sub-therapeutic doses (Annunziata et al.(2013) Invest. New Drugs. 31: 77-84). Given that different EphA2epitopes distinctly influence the kinetics and pathway ofinternalization, it is possible that the aforementioned setback with theanti-EphA2 antibody-auristatin E-conjugate is an isolated phenomenonrelating to the particular antibody used. In any event, anti-EphA2antibodies can be utilized to deliver payloads other than auristatin. Assuch there still could be further development of an anti-EphA2antibody-based therapeutic in the future. Our anti-EphA2 antibody isinternalized by the tumor selective macropinocytosis pathway, and maythus have a different potency/toxicity profile than those previouslyreported EphA2 targeting agents. Given that the macropinocytosingepitope bound by our HCA-F1 antibody is conserved across species, anytargeted therapeutics developed from this antibody can be tested insmall rodents to obtain meaningful toxicology profiles.

We have previously developed an LCM-based selection strategy to enrichfor phage antibodies binding to tumor cells in situ residing in theirtissue microenvironment as opposed to cell line artifacts (Ruan et al.(2006) Mol. Cell Proteomics. 5: 2364-2373). In this report we furtherscreened the LCM selection output using our HCA-based method andidentified novel macropinocytosing human antibodies targeting clinicallyrelevant tumor antigens. Integrating LCM and HCA into phage antibodydisplay library selection thus allows identification of novel antibodiesthat target true tumor antigens expressed by tumor cells residing intheir tissue microenvironment and enter target cells via tumor selectivepathways such as macropinocytosis. Targeted therapeutics based on thesenovel antibodies have the potential to improve potency in tumor killingand reduce toxicity on normal tissues, thus widening the therapeuticwindow and improving effectiveness of such antibody-targetedtherapeutics.

Example 2 Potent Tumor Cell Killing by a Macropinocytosing Antibody-DrugConjugate (ADC)

FIG. 18 illustrates potent tumor cell killing by a macropinocytosingantibody-drug conjugate (ADC). The macropincytosing antibody HCA-F1 wasconjugated to monomethyl auristatin F (MMAF) via amaleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vcPAB)linker. The prostate cancer cell line Du-145 cells were seeded at 1,500cells per well in 96-well plates, and incubated with the HCA-F1 ADC at37° C. for 96 hours. Viability was determined using the Calcein-AMassay. EC50, estimated by curve fit using GraphPad, is 155 pM.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

What is claimed is:
 1. An isolated antibody that is internalized into acell via a macropinocytosis pathway, wherein: said antibody comprises VHCDR1, VH CDR2,VH CDR3, VL CDR1, VL CDR2, and VL CDR3 of HCA-F1(represented by SEQ ID NOs: 2 and 6); or said antibody comprises VHCDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 of HCA-F2(represented by SEQ ID NOs: 3 and 7).
 2. The antibody of claim 1,wherein said antibody comprises VH CDR1, VH CDR2,VH CDR3, VL CDR1, VLCDR2, and VL CDR3 of HCA-F1 (represented by SEQ ID NOs: 2 and 6).
 3. Theantibody of claim 1, wherein said antibody comprises VH CDR1, VH CDR2,VHCDR3, VL CDR1, VL CDR2, and VL CDR3 of HCA-F2(represented by SEQ ID NOs:3 and 7).
 4. The antibody of claim 1, wherein said antibody is anantibody selected from the group consisting of an intact immunoglobulin,a Fab, a (Fab′)₂, an scFv, and an (ScFv′)₂.
 5. The antibody of claim 1,wherein said antibody comprises the VH domain (SEQ ID NO:2) and the VLdomain of HCA-F1 (SEQ ID NO:6).
 6. The antibody of claim 1, wherein saidantibody comprises the VH domain (SEQ ID NO:3) and the VL domain ofHCA-F2 (SEQ ID NO:7).
 7. An immunoconjugate comprising an antibody ofclaim 1 attached to an effector wherein said effector is selected fromthe group consisting of a second antibody, a detectable label, acytotoxin or cytostatic agent, a liposome containing a drug, aradionuclide, a drug, a prodrug, a viral particle, a cytokine, achelate, and an siRNA.
 8. A pharmaceutical formulation said formulationcomprising: a pharmaceutically acceptable excipient and an compositioncomprising an antibody of claim
 1. 9. The antibody of claim 4, whereinsaid antibody is an intact immunoglobulin.
 10. The antibody of claim 9,wherein said antibody is an IgG or an IgA.
 11. The immunoconjugate ofclaim 7, wherein said antibody is attached to a moiety selected from thegroup consisting of an siRNA, a cytotoxic protein, and a cytotoxicand/or cytostatic drug.
 12. The immunoconjugate of claim 11, whereinsaid antibody is attached directly or through a linker to one or more ofthe following: said cytotoxic or cytostatic drug a lipid or liposomecomplexed with and/or containing said cytotoxic or cytostatic drug; apolymeric drug carrier comprising said cytotoxic or cytostatic drug; anda nanoparticle drug carrier comprising said cytotoxic or cytostatic. 13.The immunoconjugate of claim 12, wherein said drug is an anti-cancerdrug.
 14. The immunoconjugate of claim 13, wherein said drug is selectedfrom the group consisting of a tubulin inhibitor, a DNA interactingagent, and a pathway or enzyme inhibitor.
 15. The immunoconjugate ofclaim 14, wherein said drug is selected from the group consisting ofauristatin, 1,-(2-chloroethyl)-3-cyclohexyl-lnitrosourea,1,3-bis(2-chloroethyl)-1-nitosourea (BCNU), 5- fluorouracil,5-trifluoromethyl-2′-deoxyuridine, 6-mercaptopurine, 6-thioguanine(6-TG), abraxane, abraxane, actinomycin D, anastrozole, azathioprine,belotecan, bendamustine, busulfan, camptothecin, camptothecinderivative, capecitabine, capecitabine, carboplatin, carboplatin,carmustine, chlorambucil, chloromethine, cisplatin, cladribine,colchicine, combretastatin, cyclophosphamide, cytosine Arabinoside,dacarbazine (DTIC), daunorubicin citrate, docetaxel, dolastatin,doxorubicin, epirubicin, erlotinib, etoposide, exemestane, flourouracil(5-FU), floxuridine (5-fluoro-2), fludarabine phosphate, fotemustine,gemcitabine, goserelin acetate, hexamethylmelamine, ifosfamide, imatinibmesylate, interferon, irinotecan, ixabepilone, larotaxel, letrozole,lomustine, mannosulfan, megestroltamoxifen, melphalan, methotrexate,methyl (CCNU), mitoxantrone, mTOR/PI3K inhibitor, nedaplatin, neosar,nimustine, ortataxel, oxaliplatin, paclitaxel, pamidronate disodium,pemetrexed, pentostatin, prednimustine, procarbazine HCL, raltitrexed,ranimustine, retinoic acid, a retinoic acid derivative, ribonucleotidereductase inhibitor (RNR), rubitecan, satraplatin, semustine, sorafinib,streptozocin, sunitinib, tamoxifen, taxol, temozolomide, teniposide(VM-26), tesetaxel, thiotepa, thioTEPA, topotecan, topotecan HCL,toremifene, trastuzumab, treosulfan, triaziquone, triethylene melamine,triplatin tetranitrate, trofosfamide, uramustine, vinblastine,vincristine, vindesine sulphate, vinflunine, vinorelbine tartrate, andzoledronic acid.
 16. The immunoconjugate of claim 14, wherein said drugis an auristatin is selected from the group consisting of Auristatin E(AE), Monomethylauristatin E (MMAE), Auristatin F (MMAF), vcMMAE, andvcMMAF.
 17. The immunoconjugate of claim 16, wherein said drug ismonomethyl auristatin F.
 18. The immunoconjugate of claim 13, whereinsaid drug is conjugated to said antibody via amaleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-cPAB)linker.